POLYPEPTIDES OBTAINABLE FROM SPECIES OF FASCIOLA, AND VACCINES, METHODS OF TREATMENT AND DNA SEQUENCES OF THE SAME

Abstract: Peptides obtainable from a species of Fasciola comprising the following sequence: Gln-Xaa-Xaa-Xaa-Xaa1-Xaa-Cys-Trp-Xaa-Xaa-Xaa2 wherein Xaa is any amino acid, Xaa1 is Gly or Glu and Xaa2 is Ser, Thr, Ala or Gly are disclosed. There are also disclosed vaccines and methods of treatment to reduce the spread of infection of liver fluke and DNA sequences of the polypeptides.

Description: POLYPEPTIDES OBTAINABLE FROM SPECIES OF FASCIOLA, AND VACCINES, METHODS OF TREATMENT AND DNA SEQUENCES OF THE SAME The present invention relates generally to a vaccine and more particularly to a vaccine useful in reducing spread of the liver fluke parasite and to polypeptides useful for same. The present invention also relates to a method of reducing spread of infection of liver fluke. Bibliographic details of the publications referred to in this specification are collected at the end of the description. Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification and figures are defined following the bibliography. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. cDNA clones "ICEA" and "ICEB" referred to in Australian provisional application No. PL7109 filed on 5 February, 1993 and from which the present application claims priority are re-named herein "Fhcatl" and "Fhcat2", respectively. Helminths such as trematode parasites (flukes) are a major cause of economic loss in domestic animals as well as causing serious disease in humans (Haroun and Hillyer, 1986). The major trematodes of economic or public health importance are Fasciola hepatica, Fasciola gigantica, Fasciola magna, Schistosoma bovis, Schistosoma matthei, Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Paramphistomum microbothium, Gigantocotyle explanatum, Dicrocoelium dendnticum, Eurytremapancr aticum,Paragonimuswestermani, Clonorchis sinensisand Opisthorchis viverrini (Spithill, 1992). Infection of humans by S. mαnsoni, S. haematobium, S, japonicum, C. sinensis-, O. viverrini, F. hepatica, F. gigantica and P. westermani is common in developing countries. Schistosoma spp. infect over 200 million people and cause disease by depositing parasite eggs in the tissues of the host causing a granulomatous immune response (McManus et al, 1993). The life cycles of all trematodes involve the release of eggs from adult parasites. These eggs produce a miracidia that invades the intermediate host (mollusc) leading to the eventual release of forms infective to mammalian hosts. Fascioliasis is caused by infection with the trematode parasite Fasciola hepatica This infection is of particular economic importance to industries involving ruminant animals such as sheep and cattle where fascioliasis causes wasting, death and reduced wool and egg production (Haroun and Hillyer, 1986). A vaccine that reduced parasite egg production would lead to reduced transmission from one infected host to other hosts and, in the case of Schistosoma spp., reduce human disease. However, there are currently no defined vaccines for prevention of any fluke infection (Spithill, 1992; McManus et al, 1993) and, in particular, which target worm fecundity. Until recently, methods of control relied heavily on the use of anthelmintic chemicals with only limited success. In Australian Patent Application No. 50283/90, significant progress was made in the development of a liver fluke vaccine by describing a vaccine comprising glutathione S-transferase (GST) derived from adult worms of EL hepatica. Despite the effectiveness of this vaccine, it is important to continue the development of alternative liver fluke vaccines especially those which target different aspects of the worm's life cycle. In work leading up to the present invention, an investigation was conducted into the use of helminth cathepsin proteases and in particular trematode cathepsin proteases and even more particularly cathepsin proteses, derived from Fasciola species as potential vaccine candidates. It has now been surprisingly discovered that vaccine! based on such proteases are effective in reducing worm viability and/or fecal egg counts and, therefore, worm fecundity. The vaccine also results in reduced egg viability. The vaccine of the present invention, therefore, will reduce spread of liver fluke in grazing animals since fewer viable eggs will be shed by the animal thus reducing contamination of the pasture. The subject vaccine is also of considerable environmental importance by reducing the need to spray infected pastures with potentially hazardous chemicals. It has been further surprisingly discovered that the cathepsin proteases comprise one or more hydroxylated proline residues. Accordingly, one aspect of the present invention contemplates a peptide and in particular an isolated polypeptide comprising a sequence of amino acid residues wherein said sequence includes, in a contiguous sequence, amino acid residues: Gln-Xaa-Xaa-Xaa-Xaa 1 -Xaa-Cys-Trp-Xaa-Xaa-Xaa 2 (SEQ ID NO. 23) wherein Xaa is any amino acid residue; Xaa j is Gly or Glu; and Xaa 2 is Ser, Thr, Ala or Gly; said polypeptide further characterised by its ability to induce an immune response in a host against a helminth. Preferably, the helminth is a trematode. Preferably the trematode is a species of Fasciola such as but not limited to F. hepatica, F. gigantica or F. magna. The most preferred species is F. hepatica. The Fasciola species may be in the mature state or in the newly excysted larval stage. The present invention is particularly directed to a polypeptide capable of inducing an immune response in a host which results in reduced worm viability and/ or fecal egg counts. Generally, the polypeptide is a cathepsin protease, cathepsin protease-like polypeptide or a polypeptide having cathepsin protease-like properties. A cathepsin protease or like polypeptide is characterised inter alia by an active site defined by conserved residues at three regions in the molecule. The cathepsin protease maybe in mature form or in a precursor form. The cathepsins consist of a family of sequences termed cathepsin B, H, L, S and G which vary in their degree of overall sequence identity. Preferably, the host is a mammal. Preferably, the mammal is a livestock animal such as but not limited to ovine or bovine species. Most preferably, the immune response is a protective immune response against worm fecundity. Accordingly, another aspect of the present invention contemplates a peptide cr polypeptide derived from or comprising a cathepsin protease isolatable from a Fasciola species and capable of stimulating antibody production in a suitable host against a cathepsin protease from Fasciola hepatica. Preferably, the antibody response results in reduced worm viability and/or fecal egg counts. The polypeptide of the present invention is conveniently a cathepsin protease isolatable from a species of Fasciola selected from F. hepatica, F. gigantica and F. magna and most preferably F. hepatica or a part, fragment or derivative thereof or is a fusion molecule comprising a said cathepsin protease or a part, fragment or derivative thereof and which polypeptide is capable of stimulating antibody production in a suitable host against a Fasciola species cathepsin protease. Such fusion molecules may comprise fusions of two or more of the same or different cathepsin proteases or parts, fragments or derivatives thereof, or fusions of one or more cathepsin proteases or parts, fragments or derivatives thereof with another protective molecule, such as, but not limited to, glutathione-S-transferase (GST) of E_--h-φ--J-ica, The polypeptide may be isolated from immature (e.g. newly excysted larval stage) or mature Fasciola species although the mature organism is the preferred source. For convenience, reference hereinafter to "F. hepatica" should be considered to include all species of Fasciola (e.g. F. gigantica, F. magnά). The term "polypeptide" is used in its broadest sense and for convenience includes peptides, polypeptides, proteins, glycoproteins and fusion molecules. The polypeptide is generally in isolated form or recombinant or synthetic form. When in isolated form, the polypeptide has undergone at least one purification or isolation step. Preferably, however, the isolated molecule is in a form suitable for use in a vaccine and/or represents at least 5%, preferably at least 20%, more preferably at least 35%, still more preferably at least 55-60%, even more preferably at least 75- 80% or yet even more preferably at least 90-100% of a composition relative to other components. The percentage content is conveniently measured by, for example, weight, activity, antibody reactivity or other like means. The present invention .extends to non-naturally occurring (i.e. synthetic) derivative of the subject polypeptides including derivatives which incorporate non-naturall occurring amino acid residues or chemical equivalent, homologues or analogues o naturally occurring amino acid residues. Preferably, the polypeptide has at least one proline residue modified to a 3-hydrox or 4-hydroxyproline. More preferably, the proline residue is 3-hydroxy proline Furthermore, where the polypeptide is a mature size cathepsin protease, it has molecular weight determined following SDS-PAGE of about 25-30 kDa and mor preferably about 26-28 kDa. However, the molecular weight may vary depending o whether the polypeptide is a precursor cathepsin protease or a part, fragment o derivative thereof. The peptide or polypeptide according to this aspect of the invention may be use inter alia as an active immunogen in a liver fluke vaccine. The cathepsin proteas useful in the vaccine may be a single molecule (including a fusion molecule) or ma comprise a mixture of cathepsin proteases. Where there is a mixture of protease isolated from Fasciola species (eg F. hepatica, F. gigantica or F. magna), or isolate from other helminths (e.g. trematodes) preferably at least one of said protease contains a hydroxylated proline residue but more preferably about 10-20% and eve more preferably about 20% of the cathepsin proteases carry at least on hydroxylated proline residue. Reference hereinafter to "Fasciola" species include reference to helminths in general such as trematodes and which contain the nove proteases of the present invention. Where the vaccine comprises a single cathepsi protease isolated from a Fasciola species or a fusion molecule thereof, the preferably at least one and more preferably at least 10-20% of the proline residue are hydroxylated at the 3 or 4 position, or even more preferably at the 3 positio Furthermore, the vaccine may comprise an immunogenic fragment of a cathepsi protease separately or a fusion molecule between two or more such immunogeni fragments. In addition, such an immunogenic fragment or fusion molecule may b fused to another protective moleucle, such as GST. Alternatively, the cathepsin protease may be synthesised by recombinant means in which case there may or may not be hydroxylation of proline residues. According to this embodiment, the vaccine comprises at least one recombinant cathepsin protease which may or may not carry a hydroxylated proline residue. The recombinant cathepsin protease may have an amino acid sequence substantially identical to the naturally occurring sequence or may contain one or more amino acid substitutions, deletions and/or additions thereto provided that following such alterations to the sequence, the molecule is still capable of eliciting an immune response against the naturally occurring cathepsin protease from a species o Fasciola, such as F. hepatica, F. gigantica or F. magna. Such an immune response preferably results in reduced worm viability and/ or reduced egg counts. A similar immunogenic requirement is necessary for any fragments or derivatives of the cathepsin protease whether made from the recombinant molecule or the naturall occurring molecule. Accordingly, reference herein to a "cathepsin protease" is considered reference to the naturally occurring molecule, its recombinant form and any mutants, derivatives, fragments, homologues or analogues thereof provided that such molecules elicit an immune response against the naturally occurring molecule from a species of Fasciola such as F. hepatica. The term "cathepsin protease" also extends to a fusion molecule between two or more cathepsin proteases or with other similar molecules related by amino acid sequences, as well as to fusion molecules with other protective molecules such as GST. Most preferably, the polypeptide for a Fasciola vaccine has an amino acid sequence substantially as set forth in SEQ ID NO. 2 (Figure 9A) or SEQ ID NO. 12 (Figure 9B) or SEQ ID NO. 24 (Figure 12) or an N-terminal sequence as set forth in SEQ ID NO. 21 or SEQ ID NO. 22 or having at least 40%, preferably at least 50%, more preferably at least 60%, still more preferably at least 70-80% and even still more preferably at least 90% similarity to the amino acid sequence or to a region or part of the amino acid sequence provided the polypeptide can stimulate antibodies to a cathepsin protease from a species of Fasciola such as F. hepatica. Preferably, the antibodies reduce worm viability and/or reduce fecal egg counts. Accordingly, another aspect of the present invention provides a polypeptide which: (i) is a cathepsin protease or like molecule; (ii) is isolatable from Fasciola species; and (iii) comprises an amino acid sequence having at least 40% amino acid sequence identity to all or part of the amino acid sequence set forth in SEQ ID NO. 2, SEQ ID NO. 12 or SEQ ID NO. 24. In a related aspect, the polypeptide which: (i) is a cathepsin protease or like molecule; (ii) is isolatable from Fasciola species; and (iii) comprises an amino acid sequence having at least 40% amino acid sequence identity to all or part of an N-terminal amino acid sequence as set forth in SEQ ID NO. 21 or SEQ ID NO. 22. Preferably, the helminth is a trematode as hereinbefore described and is most preferably a species of Fasciola such as F. hepatica or F. gigantica. In this context, a "part" is at least five and more preferably at least ten contiguous, amino acid residues. Preferably, the Fasciola species is F. hepatica. Preferably, the Fasciola species is in its mature form although the present invention extends to cathepsin proteases from a newly excysted larval stage parasite. Furthermore, the present invention extends to any related polypeptides having cathepsin protease-like properties such as those in the cathepsin family of proteases from other animal or plant cells which are capable of eliciting the appropriate immune response against a naturally occurring cathepsin protease from Fasciola. The present invention also extends to cDNA encoding the polypeptide of the present invention and preferably having a nucleotide sequence as set forth in SEQ ID NO. 1 (Figure 9A) or SEQ ID NO. 11 (Figure 9B) or being substantially similar to all or a part thereof. "Substantially similar" has the same meaning as above. A "part" in this context is a contiguous series of at least 15 nucleotides and more preferably at least 25 nucleotides. According to this embodiment, there is provided a nucleic acid molecule comprising a sequence of nucleotides which: (i) encodes a cathepsin protease; (ii) is isolatable from a helminth species; and (iii) hybridises under low stringency conditions to all or part of the nucleic acid sequence set forth in SEQ ID NO. 1 or 11 or to a complementary form thereof. The helminth is preferably a trematode and more preferably a species of Fasciola such as but not limited to F. hepatica, F. gigantica or F. magna. The nucleic acid molecule may be RNA or DNA, single stranded or double stranded, in linear or covalently closed circular form. For the purposes of defining the level of stringency, reference can conveniently be made to Sambrook et al (1989) at pp 387-389 which is herein incorporated by reference where the washing step at paragraph 11 is considered high stringency. A low stringency is defined herein as being in 0.1-0.5 w/v SDS at 37-45 °C for 2-3 hours. Depending on the source and concentration of nucleic acid involved in the hybridisation, alternative conditions of stringency may be employed such as medium stringent conditions which are considered herein to be 0.25-0.5% w/v SDS at ≥ 45 °C for 2-3 hours or high stringent conditions as disclosed by Sambrook et al (1989). Preferably, the Fasciola species is F. hepatica. Preferably, the Fasciola species is a mature organism although the present invention extends to Fasciola organisms in the newly excysted larval stage. The present invention further extends to a composition of matter comprising recombinant and non-recombinant forms of the cathepsin proteases of the present invention including their fragments, derivatives and the like. The present invention is predicated, in part, on the discovery that vaccination o animals with a preparation of one or more cathepsin proteases leads to a reductio in fecal egg counts resulting in a significant decrease in worm fecundity. There is also a reduction in egg viability. This has the result of reducing pastoral contamination and thereby reducing spread of infection of the parasite since less viable eggs are shed by the animal into the environment. The present invention extends to any animal capable of being infected by or carrying the parasite and is particularly directed to ruminant animals such as sheep and cattle. According to this aspect of the present invention, there is contemplated a method for reducing spread of a helminth parasite, said method comprising administering to an animal susceptible to infection with said parasite an effective amount of a polypeptide derived from or comprising a cathepsin protease isolatable from a helminth for a time and under conditions sufficient for the animal to develop antibodies to said cathepsin protease. Preferably, the helminth is a trematode and is more preferably a species of Fasciola such as F. hepatica, F. gigantica or F. magna The species of Fasciola is most preferably in the mature state. The polypeptide is preferably a cathepsin protease as hereinbefore described and may or may not have at least one hydroxylated proline residue. The vaccine may be a mixture of purified or partially purified cathepsin proteases including a product extracted, excreted, secreted or otherwise released from the helminth. Such a product is termed herein to "regurgitate" from the parasitic worm. Although not intending to limit the present invention to any one hypothesis as to mode of action, it is proposed herein that antibodies to the cathepsin protease result in reduction i fecal egg counts and, therefore, a reduction in worm fecundity. There is also reduction in viability of excreted eggs. The cathepsin protease(s) may be administered by any convenient route such as b oral, intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal suppository or intranasal administration. The present invention, therefore, provides a vaccine composition comprising an immunogenic effective amount of one or more cathepsin proteases as hereinbefore described and one or more carriers and /or diluents acceptable for veterinary use. The active ingredients of the vaccine composition comprising one or more cathepsin proteases or active immunogenic fragments thereof are contemplated to exhibit excellent activity in stimulating antibodies in the target animal when administered in an amount which depends on the particular case. The variation depends, for example, on the animal and the cathepsin protease. For example, from about 0.5 ug to about 20 mg, preferably from about 0.5 μg to about 10 mg and even more preferably from about 1 μg to about 1 mg of cathepsin protease or combined total of cathepsin proteases per animal per dose may be administered. Dosage regima may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The active compound may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, intranasal, intradermal or suppository routes or implanting (eg using controlled release molecules). Depending on the route of administration, the active ingredients which comprise one or more cathepsin proteases may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said ingredients. For example, the low lipophilicity of the cathepsin protease will allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis. In order to administer the vaccine by other than parenteral administration, the cathepsin protease will be coated by, or administered with, a material to prevent its inactivation. For example, the cathepsin protease may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon or other cytokines. Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Conveniently, the adjuvant is Freund's Complete or Incomplete Adjuvant. Enzyme inhibitors include pancreatic trypsin inhibitor diisopropylfluorophosphate (DEP) and trasylol. Liposomes include water-in-oil-in water CGF emulsions as well as conventional liposomes. The active compounds may also be administered in dispersions prepared in glycerol liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinar conditions of storage and use, these preparations contain a preservative to preven the growth of microorganisms. The pharmaceutical forms suitable for injectable use include sterile aqueou solutions (where water soluble) or dispersions and sterile powders for th extemporaneous preparation of sterile injectable solutions or dispersion. In all case the form must be sterile and must be fluid to the extent that easy syringability exists It must be stable under the conditions of manufacture and storage and must b preserved against the contaminating action of microorganisms such as bacteria an fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liqui polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersio and by the use of surfactants. The prevention of the action of microorganisms ca be brought about by various antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid, thormerosal, and the like. In man cases, it will be preferable to include isotonic agents, for example, sugars or sodiu chloride. Prolonged absorption of the injectable compositions can be brought abou by the use in the compositions of agents delaying absorption, for example. Sterile injectable solutions are prepared by incorporating the active compounds i the required amount in the appropriate solvent with various of the other ingredient enumerated above, as required, followed by filtered sterilization. Generally dispersions are prepared by incorporating the various sterilized active ingredient(s into a sterile vehicle which contains the basic dispersion medium and the require other ingredients from, those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof. When the cathepsin protease or mixture thereof is suitably protected as described above, the vaccine may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of .the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in the vaccine compositions is such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared,so that an oral dosage unit form contains between about 0.5 ug and 20 mg of active compound. The tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum gragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, an material used in preparing any dosage unit form should be pharmaceutically pur and substantially non-toxic in the amounts employed. In addition, the activ compound maybe incorporated into sustained-release preparations and formulations Conveniently, the vaccine is administrable with the animal feed, such as grain. Th vaccine composition may be incorporated into a grain base or may be topicall applied to feed grain. As used herein "carriers and/or diluents suitable for veterinary use" include any an all solvents, dispersion media, aqueous solutions, coatings, antibacterial an antifungal agents, isotonic and absorption delaying agents, and the like. The use o such media and agents for pharmaceutical active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the activ ingredient, use thereof in the vaccine compositions is contemplated. Supplementar active ingredients can also be incorporated into the compositions. The latter i particularly contemplated as far as the present invention extends to multivalen vaccines or multi-component vaccines. The vaccine composition of the present invention may comprise in addition to on or more cathepsin proteases, one or more other active compounds such as othe antigens obtainable from liver fluke or other parasites, immune enhancing agent and/or medicaments for veterinary use. The vaccine composition may also contai more than one variety or family of protease or may contain a partially purified E hepatica regurgitate. The vaccine may alternatively contain or may additionall contain a fusion molecule between two or more cathepsin proteases or immunogeni fragments thereof, or between a cathepsin protease or an immunogenic fragmen thereof and another protective molecule such as GST. The cathepsin protease or antigenic fragment thereof may also be delivered by a liv delivery system such as using a bacterial expression system to express the cathepsi protease in bacteria which can be incorporated into gut flora. Alternatively, a vira expression system can be employed. The present invention extends to antibodies t the cathepsin protease or antigenic fragments thereof. Antibodies may b monoclonal or polyclonal and are useful, for example, in purifying cathepsi proteases or related molecules from Fasciola species such as from F. hepatica or E_ gigantica. The present invention extends to a method of purifying a cathepsin protease o related molecule from a helminth, preferably a trematode and more preferably Fasciola species e.g. F. hepatica or from another biological sample comprisin applying a regurgitate of the helminth or applying the biological sample to an affinit column comprising a cathepsin protease reactive antibody or other suitable ligand. The column effluent can be further processed by, for example, SDS-PAGE and/ or ion exchange chromatography. Alternatively, a regurgitate or other biological sample is concentrated by ultrafiltration and subjected to molecular sieving and the material with the desired molecular weight removed. Yet another aspect of the present invention contemplates a diagnostic assay for helminth infection of an animal said method comprising screening for antibodies to the cathepsin proteases as hereinbefore described. The assay may alternatively involve screening for cathepsin protease antigens using monoclonal or polyclonal antibodies. The most convenient form of test is an ELISA but the present invention extends to any suitable form of assay. The present invention is further described by reference to the following non-limiting Figures and /or Examples. In the Figures: Figure 1 is a photographic representation demonstrating cathepsin proteinase activit in adult F. hepatica. A 50 fold concentrate of adult regurgitate (track a) and neat adult regurgitate (track b) were resolved by SDS-PAGE and the proteins visualised by silver staining. Proteolytic activity of neat adult regurgitate was detected in gelatin SDS-PAGE gels on the addition of dithiothreitol (2mM DDT) in lane (d) but only weakly without DTT (lane c). Figure 2 shows molecular sieving of the regurgitate of adult F. hepatica worms. Approximately lmg of concentrated regurgitate was passed down a gel permeation column (Example 1). Panel A: Chromatographic profile of the elution monitored at 280nm (1.0 AUFS). Peaks are numbered according to order of elution. The area of collection of one minute fractions of peaks 5 and 6 is indicated. Panel B: SDS- PAGE analysis (followed by silver staining) of the one minute fractions of peaks 5 and 6. 5% of each fraction was loaded in reducing sample buffer. Panel C: Western blot analysis (using ovine F. hepaticacat epsin proteinase antiserum) of the fractions, loading 1% of each fraction in reducing sample buffer. Figure 3 shows anion exchange chromatography of the cathepsin proteinases of the regurgitate of adult F. hepatica Material from peaks 5 and 6 from Figure 2 was pooled, concentrated and applied by FPLC to a Mono Q column in 20 mM ethanolamine (pH 9.0; Buffer A). The column was eluted with buffer B (buffer A + 2M NaCl) delivered by a discontinuous gradient which was held at 5.5% and 13% buffer B. Panel A: Chromatographic profile of the elution of proteins from the Mono Q column. Elution was monitored at 280nm (1.0 AUFS) and 13 fractions were collected as indicated. Panel B: SDS-PAGE analysis of fractions 1-13 followed by silver staining. Approximately 1% of each fraction was loaded in reducing sample buffer. Panel C: Western blot analysis (using the ovine F. hepatica cathepsin proteinase antiserum) of fractions 1-13. Approximately 1% of each fraction was loaded in reducing sample buffer. Figure 4 is a photographic representation of a gel analysis of Mono Q fractions of cathepsin proteinases under non-reducing conditions. Approximately 1 % of fractions 2-5 from Fig 3A was loaded onto the gels and separated by non reducing SDS- PAGE. Proteins in the SDS-PAGE gel were detected by silver stain and the cathepsin proteinases were detected by the ovine antiserum in the Western blot. Figure 5 is a photographic representation of SDS-PAGE analysis of the purified cathepsin proteinases under non-reducing (NR) and reducing (R) conditions. An aliquot of lOμg of protein was loaded in each lane and visualised by silver stain. Figure 6 is a graphical representation of ELISA analyses of antibodies to cathepsin proteinases of F. hepatica during the course of the trial. Individual sera from bleeds at weeks -6 (first immunisation), -2 (boost), -1 (one week prior to challenge), and weeks 4 and 12 post-infection were measured by ELISA for anti-cathepsin proteinase activity (see Example 1). Panel A follows the development of anti-cathepsin proteinase titres in the infected control sheep. A maximum titre of 10,000 was achieved by one animal at week 12. Panel B monitors the development of anti- cathepsin proteinase titres in the immunised sheep over the 18 weeks of the trial. A maximum titre of 225,000 was obtained by one animal at week 12. Figure 7 is a photographic representation showing two dimensional SDS-PAGE analysis of purified excretory/secretory cathepsin proteases of adult F. hepatica Isoelectric focussing in the first dimension was conducted over a pH range of 2.5-7.0 with 30μg purified proteases. Proteins were detected in the second dimension (15% w/v SDS-PAGE) by silver stain. The lane (R) is a single dimensional reducing SDS- PAGE run of the cathepsin protease preparation. Figure 8 is a graphical representation showing pH optima of the excretory/secretory cathepsin proteases of adult F. hepatica Total enzyme of 15 and 30ng was added to lOOmM Tris and lOOmM phosphate buffers, respectively. The assay was performed over the pH range indicated using the fluorogenic substrate, Z-FR NMeC, as described in Example 2. Figure 9A: Peptide alignment with the predicted amino acid sequence and the nucleotide sequence of Fhcatl. The nucleotide sequence of the Fhcatl (ICEA) cDNA is 1075 bases in length. The predicted amino acid sequence begins at methionine (nucleotide 25) and ends at phenylalanine (nucleotide 1002). The direct N-terminal sequence .(N-term) is also mapped to the Fhcatl primary sequence Peptides were derived from chymotryptic (CT) and endo-Glu-C (GC) digests o purified F. hepatica excreted cathepsin proteases. Figure 9B: Representation showing the nucleotide sequence and predicted amino aci sequence of another cathepsin protease (Fhcat2 [ICEB]) from liver fluke. Figure 10: Alignment of the amino acid sequences of the Fhcatl preproprotein human preprocathepsin L (HUMCATL: P07711), bromelain (P14518), huma preprocathepsin H (HUMCATH: P09668), human preprocathepsin B (HUMCATB P07858),and a ScΛistasσmα thiol cathepsin (SchistoB: N21309). (Genbank accessio numbers are indicated within the brackets). Functionally conserved amino acids ar boxed to show the similarity among these sequences. Figure 11: N-terminal sequences of the excretory/secretory cathepsin proteinases o adult F. hepatica Sequences were generated either by direct N-terminal amino aci sequencing or from peptides isolated from an endoGlu C digest of the same materia (Material and Methods). Alignments were found to the N-termini of bromelai (Ritonja et al 1989) and papain (Drenth et al. 1971), several mammalian thio cathepsins (bovine cathepsin S (Wiederanders et al.1991), mouse cathepsin B (Cha et al. 1986), human and mouse cathepsin L (Mason et al. 1988; Joseph et al, 1988 and to Trypanosoma cathepsin proteinases (Mottram et al.1989, Eakin et al.1992) Figure 12: Amino acid sequence of the FhcatBl sequence and alignment with Huma Cathepsin B; Schistosoma mansoni Cathepsin B; Human Cathepsin L; Fasciol hepatica Cat 1 & 2. Gaps in the Fhcat BI sequence indicate sequences to b confirmed. Bolding indicates identical residues between the sequences. EXAMPLE 1 DEVELOPMENT OF VACCINE 1. Materials and Methods Parasites: Metacercariae were maintained by passage through the intermediate host snail Lymnaea tomentosa The original isolate derived from an infected sheep in Lancashire, U.K., (designated herein as "Compton 2") was purchased from Compton Paddock Laboratories (Surrey, U.K.) and is maintained by passage through the local snail host and Merino sheep as the mammalian host. Gel analysis: Various preparations of regurgitate and its extracts were analysed in reducing conditions on 15% w/v SDS-PAGE (Laemmli, 1970) and the proteins visualised by silver stain (Morrissey, 1981). Proteinase activity in adult regurgitate was visualised by gelatin gels (Dalton & Heffernan, 1989). Briefly, gels were prepared as for normal SDS-PAGE (Laemmli, 1970) except for the co-polymerisation of gelatin (0.1% w/v) in the resolving gel. Prior to electrophoresis, the gels and running buffer were cooled to 4 °C to reduce enzymatic activity during the run. Samples were mixed 1:1 with non-reducing SDS- PAGE sample buffer and loaded onto gels without denaturation. After electrophoresis, the gels were incubated in two 30 minute changes of 0.1 M sodium citrate (pH 4.5) containing 2.5% v/v Triton X-100, followed by a final incubation in 0.1M sodium citrate (pH 4.5) (containing 2mM dithiothreitol (DTT) when appropriate.) for 1 hour at 37 °C. Gels were stained with 0.1% w/v Coomassie Blue (in 50% v/v methanol, 40% v/v acetic acid, 10% v/v distilled water) for 15 minutes and then destained in 7% v/v acetic acid. Western blots were prepared as described (Burnette 1981), electrotransfers being conducted in a Biorad Minitransblot Module over 90 minutes at 150V at 4 °C in precooled (-20 °C) transfer buffer. The blotted nitrocellulose filters (Scheicher and Schuell, Dassel, West Germany) were blocked in 5% Blotto-PBS/Tween for 1 hour. After washes in PBS/Tween, antisera diluted in PBS/Tween were applied to filters for 18 hours (on a rocking platform) at 4 °C. Following further washes, species specific Ig horseradish peroxidase conjugates (Silenus, 1:400 in PBS/Tween) were added to the blots for 2 hours (RT), and the enzyme-immunocomplexes finally visualised by reaction with 4-chloro-l-naphthol (Sexton et al., 1990). The ovine antisera to F. hepatica cathepsin proteinase was prepared as described below. The rabbit anti-bromelain serum was obtained from the Victorian Institute of Animal Science, Attwood, Victoria 3049, Australia. Antigen Preparation: Fasciola hepatica cathepsin proteinases were purified from the regurgitate of mature worms. Adult fluke harvested from the livers of sheep infected with the Compton 2 strain, were washed three times in warm (37 °C) PBS and were incubated for 2-4 hours in Basal Eagles medium (Flow Laboratories, Melbourne) supplemented with lOmM glutamine (Flow Laboratories) and buffered with 0.06% w/v NaHCθ3 (pH 7.5) at 37 °C in 5% v/v C0 2 . The medium was then decanted and frozen (-20 °C) till required. On thawing, the regurgitate was clarified by centrifugation (10,000g, 30 minutes, 4 °C), vacuum filtered on a 0.45 μ filter, and then Ultrafiltered to a 40-50 fold concentrate (Minitan Ultrafiltration System, Waters-Millipore, Bedford, MA) using a pre-equilibrated 10 kDa cut-off membrane (Minitan Plates: NMWL, Millipore, Bedford, MA) for 8 hours at 8psi. During ultrafiltration reservoirs were kept on ice. Typically, 2 1 batches were concentrated to 40-50ml retentate. Due to batch to batch differences in protein concentration, more dilute retentates compared to other retentates were further concentrated by vacuum centrifugation (Savant Instruments, Hicksville, NY). Molecular sieving was conducted on a precalibrated Superose 6 column (Pharmacia) by FPLC (Pharmacia). The column was pre-equilibrated by an overnight wash in 0.6M NaCl, lOmM Tris (pH6.0) and samples for separation were dialyzed overnight in this buffer. Aliquots (lmg) of the concentrated regurgitate were microcentrifuged and filtered before delivery on the column by FPLC at 0.4ml/min. Runs were conducted over 80 min using a 280nm detector to monitor elution. Material eluting under the 30kD peak was collected, pooled and in preparation for anion exchange was dialyzed against water (overnight, 4 °C) to reduce the salt content and then concentrated 5-10 fold by vacuum centrifugation (Savant). This concentrate was further dialysed into 20mM ethanolamine (pH 9.0) overnight at 4 β C. A Mono Q column (Pharmacia) was also pre-equilibrated overnight in the same buffer. Subsequent to microcentrifugation and filtration, up to 5ml of the dialysate was loaded onto the anion exchange column. Elution of the protein components occurred during a programmed run which allowed for a 5 minute isocratic step (in Buffer A: 20 mM ethanolamine (PH 9.0)) and then a programmed gradient which delivered salt onto the column at 14mmol/min from buffer B (Buffer A + 2M NaCl). At 5% buffer B, the gradient was paused to allow elution of the first major peak, and then returned to normal program. Material in the first peak eluted from the ion exchange runs was dialyzed against distilled water and concentrated 5 fold by vacuum centrifugation and then further dialysed. A radioiodinated tracer of this material was prepared by the Chloramine T method (Sonda and Schalamonit 1970). The radiolabelled tracer was mixed with the dialysate to give a specific activity of 5-10 x 10 4 cpm/μg protein. The mixture was denatured in non-reducing SDS-PAGE sample buffer and loaded onto preparative 15% w/v SDS-PAGE gels. On completion of the run, the gels were dissembled, wrapped in plastic, fluorogenic markers added, and exposed to X-ray film (XAR-5, Eastman-Kodak, Rochester, NY) for 30-60min at RT. Using the markers as a guide, the 26 kD radioactive band was excised, fragmented and boiled in 1% w/v SDS in lOmM Tris (pH 8.0) for 30 min. The suspension was diluted to 0.1% w/v SDS and then rotated overnight at RT. After allowing the contents to settle, the supernatant was removed, and the gel pieces washed in 0.1% w/v SDS in 10 mM Tris (pH 8.0). Gel pieces were then resuspended in the same buffer and eluted for a further 24 hours. This second eluant was pooled with the first eluant and then precipitated with 9 volumes of acetone at -20 °C. The pelleted precipitate was resolubilised in 200μl 1% w/v SDS, 20 mM Tris (pH 8.0) and pooled with resolubilised precipitates from other gel runs. The pooled material was acetone precipitated. As all the contents from a batch of concentrated regurgitate was processed, all acetone precipitates were resolubilised in 1% w/v SDS, lOmM Tris (pH 8.0) and then precipitated with volumes of ethanol. The pelleted precipitates were resolubilised in 100% TCA and diluted to 10% v/v TCA on addition of water (4 °C) and left on ice for 1 hour. The TCA pellets were twice washed in acid- ethanol and then ethanol to remove the last traces of salt and SDS. Samples of this material were finally checked for purity and the protein content estimated on non- reducing SDS-PAGE gels which were silver stained. ■Antibody Production- Antisera to the cathepsin proteinase was produced by immunising 5 Merino wethers with approximately 70μg of apparently pure proteinase generated from adult regurgitate. For this preparation of antigen, the purification procedure was based on that of Coles and Rubano (1988). Regurgitate (from 8-48 hour cultures of adult fluke) was clarified by centrifugation and chilled. Cold ethanol was added dropwise (2.2 ml/min) into mixing chilled regurgitate (300-400ml at 0-2 °C), until a final ethanol concentration of 60%v/v was achieved. The solution was equilibrated at - 20 °C for 18 hours and then pelleted at 6,300g (30min, 4 °C). The supernatant was then taken up to a final concentration of 75% v/v ethanol, equilibrated overnight at - 20 °C, and clarified (as above). The pellet was washed in 100% ethanol and resuspended in water. This material was examined by silver stained SDS-PAGE for a complex of bands at 28kD known to contain cathepsin proteinase activity (Coles and Rubano, 1988). If the preparation was deemed impure due to the presence of other species, the material was subject to gel purification with the addition of a radiolabel trace of apparently pure material. These steps were performed similar to that described in the previous section. Ten such preparations were completed to yield of approximately 500μg of material containing only the 28kD complex. For primary immunisation approximately 250μg protein was homogenised with Freund's Complete Adjuvant (FCA)(Commonwealth Serum Laboratories, (CSL) Melbourne, Australia) to be subcutaneously (s.c.) injected into five Merino wethers. Approximately 30μg of 28kD protein was injected. A second immunisation with 40μg of the purified 28kD material in Incomplete FCA (CSL) occurred 4 weeks later. Sera from these animals were collected after a further 4 weeks and pooled in equal volumes to be used as a reagent to detect the presence of cathepsin proteinases. Trial protocol: Merino wethers (5 year old) were obtained from farms free of liver fluke in New South Wales (Australia). Individual animals were screened for F. hepatica eggs in their faeces and for low serological cross reactivity to a F. hepatica extract as analysed by ELISA (described below). Ten animals were immunised (s.c.) with 120μg purified cathepsin proteinase in water, mixed with an equal volume of FCA (CSL) six weeks before infection (week -6). Four weeks later (week -2), this same group was boosted (s.c.) with 90μg of purified antigen mixed with Incomplete FCA (CSL). These animals and a further 10 unimmunised controls were challenged by intraruminal delivery of 300 metacercariae in water two weeks after the boost (week 0). Animals were maintained on open paddocks which were free of liver fluke. Blood samples were taken at weeks -6 (prebleed and immunisation), -2 (boost), -1, 0 (challenge), 4, and week 12. Animal health was monitored by assessment of parasite induced anaemia by estimation of red blood cell haemoglobin (van Kampen and Zijlstra, 1961) determined on a Cobas MIRA automatic analyser (Roche, Basel, Switzerland). Sera were collected and stored at -20 °C till analysed. At 14 weeks post challenge, fecal samples were collected from all sheep which were then humanely slaughtered to obtain the livers and gall bladders. Worms were collected from the liver tissues, bile ducts and gall bladders. The egg mass from the gall bladder were also collected. Fecal egg counts (FEC) were performed as described (Sexton, et al. 1990). Serciogyr Animals were examined for prior exposure to F. hepatica or any other parasite th would elicit high levels of crossreactive antibodies by ELISA analysis of seru antibodies to a whole Fasciola extract. In this assay, polyvinyl microtitre plate (Immuno-Maxisorp plates, Nunc, Denmark) were coated overnight at 4 °C with 1/2000 dilution of adult worm lysate (Wijffels, et al., 1992) in coating buffer (0.1 Na 2 C0 3 , pH 9.5). Sheep sera were initially diluted to 1:500 in Blotto-PBS /Twee (5% w/v skim milk powder, 0.05% v/v Tween 20 (Sigma) in PBS, pH 7.2) an titered out on the plate by serial 2-fold dilution. After an incubation of 1 hour 37 °C, plates were washed in PBS/Tween and a Donkey anti-sheep Ig horse radis peroxidase conjugate (Silenus, Melbourne, Australia) diluted 1:2000 in Blott PBS /Tween was applied for 1 hour at 37 C C. Subsequent to a further wash an addition of substrate solution (ImM 2,2-azinobis(3-ethylbenathiazole sulphonic aci (ABTS), Sigma) in 62mM citric acid/76 mM Na2HPO pH 4.0, supplemented wit 0.03% v/v H2O2), colour development was measured at 414nm using and automate Titertek Multiskan spectrophotometer (Flow Laboratories). Animals with hig serum titres (3-5 fold above the mean) were rejected for use in the trial whic usually equated to 10% of any sheep population with no history of exposure t F. hepatica. Throughout the trial development of antibodies to F. hepatica cathepsin proteinase was monitored by ELISA. Antigen from a very dilute solution (1:16,000 in coatin buffer) of adult regurgitate concentrate was adsorbed onto microtitre plates (Nun overnight (4 °C). After a 1 hour blocking step (Blotto-PBS /Tween at RT), sera wer titered out from 1:1000 by 2-fold dilution (in PBS/Tween) and incubated overnig at 4 °C. The anti-sheep Ig conjugate was applied for 4 hours at RT prior to reado with ABTS. Titres were determined by direct comparison to individual preblee which were similarly assayed over the same dilution range. Titres were determine as the highest serum dilution with 0.1 absorbance unit above the same dilution of th prebleed. Concentrated and ultrafiltered regurgitate was used as the antigen in preference to untreated regurgitate due to the detectable presence of host (sheep) immunoglobulin fragments in this latter material. Presumably, ultrafiltered regurgitate contained reduced levels of these fragments. Furthermore, the use of purified cathepsin proteinase as antigen revealed no advantage in sensitivity or specificity over the regurgitate concentrate in this assay. Egg Viability: The contents of the gall bladder were collected and stored at 4 °C in Alfoil-covered glass containers until samples could be further processed. Eggs were separated from the bile contents by several washes in tap water. A final suspension in water was incubated (in the dark) at 27-29 °C for 14 days. The suspensions were then exposed to an incandescent light source for 20 minutes. To assess viability, 10 drops of 1% w/v Lugol's iodine solution was added to a 25ml sample of the suspension, and the contents assessed under microscope for hatched miracidia and for viable eggs (i.e. those with eye spots); eggs with no sign of differentiation were considered as not viable. 2. Preparation of a Cathepsin Protease Vaccine Cathepsin proteinases identified in the regurgitate of mature F. hepatica worms. SDS-PAGE analysis of total adult liver fluke regurgitate followed by silver staining revealed a distinct complex of 2-3 bands at approximately 28-30 kDa (Fig. 1, tracks a and b). It was considered possible that this complex or a component of it was responsible for the proteolytic activity detected in the same preparation at a similar position in the gelatin gel (Fig. 1, tracks c). This gelatinolytic activity is greatly enhanced by addition of dithiothreitol which suggested the presence of a cathepsin proteinase (Fig 1, track d). The putative cathepsin proteinases were purified from concentrated regurgitate using several chromatographic procedures. A fifty-fold ultrafiltration concentrate was applied to a gel permeation column in a high salt Tris buffer (pH 6.0) and partially resolved into 5 major peaks (peaks 1-5) and a minor late eluting peak (peak 6, Fig. 2A). Use of the high salt buffer and slightly acidic pH allowed best separation of peaks 5 and 6 from the earlier eluting peaks. Reducing SDS-PAGE analysis of one minute fractions of peaks 5 and 6 revealed the presence of a 28kD component in all fractions (Fig. 2B). Clearly fractions from peak 5 rather than peak 6 contained far more of the 28kD species. Lower molecular weight species ( - 10-14 kD) were also detected in the early fractions (1-5), some of which may represent contaminating proteins from peak 4. Western blot analysis of the fractions from peak 5 and 6 using the ovine antiserum to cathepsin proteinase revealed an immunodominant species at 28kD in all fractions and the most intense staining coincided with fractions from peak 5 (Fig. 2C, tracks 1-5). Lower molecular weight species at 17kD, 15kD and 14kD were also detected by the antiserum in most of {he fractions although they were less easily discerned in the later fractions, probably due to less material in these fractions. It was assumed that these species represented breakdown products of d e cathepsin proteinases, and appeared to coincide with the lower molecular weight species visualized in Fig. 2B. As there was no apparent difference in the antibody reactive material under peaks 5 and 6 (Fig. 2) these fractions were pooled and, after appropriate dialysis and concentration, were subjected to anion exchange chromatography. Holding the salt gradient at 5.5% buffer B (lOmM NaCl in 50mM ethanolamine (pH 9.0)) resulted in elution of a major group of proteins well separated from higher salt eluting components (Fig. 3A). SDS-PAGE analysis revealed that the first major cluster of 5-7 peaks contained very little protein at the 28 kD region and the most intense staining appeared at 14 and 15 kD region (Fig. 3B tracks 1-5). However, Western blot analysis of these fractions detected components at 17, 15 and 14 kD which were reactive to the cathepsin proteinase antiserum (Fig. 3C) and was consistent with the material collected from the gel permeation step (Fig. 2C). The lower molecular weight species appeared to represent die breakdown products of the cathepsin proteinases. However, the difference in staining intensity of the 28 kD species and other components between these two techniques seemed incongruous and warranted further investigation. When the material in fractions 2-5 of the major cluster of anion exchange peaks (Fig 3A) was resolved on non-reducing SDS-PAGE gels and visualized by silver staining, low molecular weight species at 14 and 15 kD were still detected although the 26-28 kD region was staining more intensely (Fig. 4 left panel). This result suggested the presence of far more protein in the 28 kD region of the gel than had been indicated by the reducing SDS-PAGE analysis (Fig. 3B). However, when the non-reduced preparation was examined by Western blot the 14 and 15kD species were not found to be antibody reactive and only species at 26kD and larger were now detected with the antisera. It was apparent that a contaminating protein had co-eluted with the cathepsin proteinases in both the molecular sieving and anion exchange procedures so that in reducing SDS-PAGE gels, the contaminant appeared to give rise to products that were obscured by fragments of the reduced and denatured cathepsin proteinases. The shift in migration of the major form of the cathepsin proteinases between reducing and non reducing SDS-PAGE gels suggests internal disulphide bonds in which some forms of the proteinases give rise to lower molecular weight products on reduction. To ensure that the cathepsin proteases were purified, the major eluting peak from the ion exchange runs was dialysed, concentrated and separated by preparative SDS- PAGE in non-reducing conditions using a trace label. Vaccination trial with purified cathepsin proteinase cfF. hepatica. Ten animals were immunised (over two injections 4 weeks apart) with a total of 210μg of cathepsin proteinase purified from adult F. hepat ica regurgitate. A control group of 10 sheep were not immunised. Two weeks later both groups were challenged with 300 metacercariae of F. hepatica delivered by intraruminal injection. Development of serum antibodies to fluke cathepsin proteinases was monitored by ELISA. As seen in Fig. 6 (Panel A), all individuals in the infected control group generated low levels of antibodies to the cathepsin proteinases 12 weeks post infection. The specific antibody profile of the immunised animals appropriately detected the primary and secondary responses to immunisations (Fig. 6, Panel B). At four weeks post-challenge, nine out of ten individuals in the immunised group had increased antibody titres to the Fasciola cathepsin proteinases; half the group showed a marked increase. Late into infection, at week 8, these antibody levels were sustained or increased in 8 out of 10 animals. Worm Fecundity Table 1 summarises the worm burdens and final FEC of the trial animals. The control group returned worm burdens over the disperse range of 34-99 adult worms, causing an arithmetic mean of 69.6 and a large coefficient of variation (c.v.) of 33%. The spread of worm burden data was even more severe for the immunised group, yielding an average worm count of 79.3 adult fluke per sheep with a standard deviation of 42 (c.v. = 53%). Clearly, immunisation with the fluke cathepsin proteinases under the regime used does not reduce burdens after 14 weeks of infection with F. hepatica metacercariae. However, FEC were markedly affected in the vaccinated group. Average fecal eggs /gram faeces excreted in this group (206 ± 86) were 70% reduced as compared to controls (700 ± 377) and this difference was statistically significant (p < 0.05) using Tukey's multiple range test. As with the worm burden data, coefficients of variation were large, 54% and 42% for the control group and the vaccinates respectively, but there was littie overlap in the spread of the two groups. When worm burdens and FEC are taken together to determine egg/gram faeces per worm, the vaccinates show a 67% reduction in worm fecundity. However, the large variances of these incorporated parameters cause a correspondingly large c.v. in the worm egg output figures, 68% and 56% for controls and vaccinates, respectively. Arithmetic means were 11.1 egg/g/worm for controls compared to 3.10 egg/g/worm from the vaccinated group. This difference was significant (p < 0.05) using Tukey's multiple range test. Egg Viability Eggs from the gall bladder were collected and assessed for viability (Table 2). Comparisons made between the controls and vaccinates show that with an average egg viability in the vaccinates of 38% ± 39%, there is a reduction of 56% in egg viability from this group. EXAMPLE 2 ANALYSIS OF CATHEPSIN PROTEASES IN VACCINE OF EXAMPLE 1 1. Materials and Methods Gel -Analyses: Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970) analyses were carried out on 15% w/v gels which were silver stained (Morrissey 1981). For two dimensional gel analysis, 30μg of the purified cathepsin proteases was applied to an isoelectric focusing gel (OTarrell, 1975) in the first dimension (incorporating Pharmacia ampholytes pH 2.5-5 and pH 5-7 mixed at a ratio of 1:2). SDS-PAGE was conducted in the second dimension using 15% reducing conditions and the gel silver stained (Morrissey, 1981). Western blot analysis using an ovine cathepsin protease antiserum was conducted as previously described in Example 1. Whole fluke lysates were prepared as described Wijffels et al. (1992). Purification cfF. hepatica cathepsin proteases. The cathepsin proteases were essentially purified from adult liver fluke regurgitate by the procedure described in Example 1. Production of Peptides Peptides were generated from several digests of purified F. hepatica cathepsin proteases. Several peptides were produced from a digest using endoproteinase-Glu- C (endo-Glu-C; Boehringer-Mannheim, Germany). Peptides GC3.1, 15.2, 20.2 and 21.2 were products of a digest of the cathepsin proteases purified as described above. Approximately 20μg of the cathepsin protease preparation was S-pyridylethylated and coprecipitated (in acetone) with 2% w/v endo-Glu-C. Digestion was performed at 37 °C for 9 hours in lOOμl of 0.1M NH4CO3 (pH 8.0, C0 2 ). On completion of digestion the mixture was dried down by vacuum centrifugation and resolubilised in 6M guanidium HCl (in lOmM Tris (pH 8.0)) and injected onto a C18 Nova-Pak reverse phase column (Waters-Millipore, Milford, MA). Peptides were resolved with a 5-60% acetonitrile (AcN) gradient (in 0.1% trifluoroacetic acid (TFA)) delivered by HPLC at 0.5 ml/min (Waters 625 LC System). Elution was monitored at 214nm and high absorbance peaks collected by a timed loop. The contents of selected peaks were repurified on the same system using a 0-40% AcN (in 0.1% TFA) gradient. To obtain larger peptides, a chymotryptic digest was performed on lOOμg of purified protein that had not been reduced and alkylated. Digestion was performed over 4 hours but otherwise was conducted as described Wijffels et al. (1992). Ensuing peptides were purified and refractionated on a C8 Nova-Pak (Millipore-Waters) reverse phase column using 5-60% AcN (in 0.1% TFA) gradient delivered by HPLC at 0.5 ml/min. This digest yielded the chymotryptic peptides CT13.2 and CT11.3. Two other chymotryptic peptides, CT21.2 and CT13.3 were obtained from a digest of the same preparation of cathepsin proteases described in Example 1. The chymotryptic digest was performed as previously described (Wijffels et al. 1992). Peptides were purified as for the endo-Glu-C digest. Purified peptides were dried by vacuum centrifugation prior to N-terminal sequencing conducted by a Model 471 A Protein Sequencer (Applied Biosystems Inc. (ABI), Foster City, CA). PTH-amino acids were resolved by a gradient of 5% tetrahydrofuran (in 50.1 mM sodium acetate (pH 3.9)) and 100% AcN (ABI) delivered by HPLC at 1 ml/min onto a Spheri-5 isoPTH reverse phase column (ABI). Enzym tic Assays Solutions of purified cathepsin proteases were assessed for proteolytic activity by a fluorogenic assay based on that of Barrett (1980). Reactions proceeded in a 1ml volume with final concentrations of 250μM substrate, 2mM DTT, lOOmM buffer and 0.05% v/v Brij 35 with 15-50ng total purified enzyme. The mixture was incubated for 25 minutes at 37 °C, and immediately followed by addition of 1ml 0.1% v/v Brij 35 and fluorescence determined by a fluoresence spectrophotometer (Perkin Elmer Model MPF-3) set with emission and excitation wavelengths of 352 and 440nm respectively. To determine the inhibitory effect of various agents, the enzyme was preincubated with buffer, DTT and inhibitor for 10 minutes at RT prior to addition of substrate. Substrates (N-CBZ Phe- Arg 7-amino-4-methyl coumarin (Z-FR NMeC) and N-CBZ arginine 7-amido-4-methyl coumarin (Z-R NMeC)) and inhibitory reagents (phenyl methyl sulphonyl fluoride (PMSF), iodoacetamide (IAA), leupeptin, antipain, aprotinin, and E-64 (trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane)) were all purchased from Sigma with the exception of EDTA which was obtained from BDH. Immunosσeening of cDNA libraries A λZAP F. hepatica cDNA library was plated on a lawn of Escherichia coliB 4 cells at a density of 50,000 plaque-forming units (pfu) per 150mm L-Broth agar plate. Approximately 5 x IO 5 pfu were screened for expression of ICE of F. hepaticaυsing the Protoblpt method as described in the Protoblot Technical Manual purchased from PROMEGA (Madison, USA). The libraries were screened with a pooled sheep antiserum raised to the purified ICE of adult F. hepatica at a dilution of 1/600. Filters were blocked in a buffer containing lOmM Tris HCl, pH8.0, 150mM NaCl, 0.05% v/v Tween 20, 1% w/w gelatin. Positive plaques identified in a primary screen were picked, replated at a lower density and rescreened with the ovine antiserum until individual positive plaques were identified. Isolation and sequencing af cDNA inserts Phagemid DNA containing cDNA inserts from positive λZAP phage clones was isolated by excision in vivo of the pBluescript phagemid under the conditions recommended by Stratagene (La Jolla, USA). Phagemid DNA was extracted by the method of Birnboim and Doly (1979). DNA sequencing of cDNA inserts was performed by the chain termination method (Sanger et al. 1977) after the plasmid DNA was denatured by treatment with NaOH. 2. Analysis of Cathepsin proteases Heterogeneity af cathepsin proteases of F hepatica The heterogenous nature of the excreted /secreted cathepsin proteases of F. hepatic was initially observed in the gel permeation profiles. Under conditions of high salt and slightly acidic pH, the majority of the cathepsin proteases elute under one peak with a small proportion eluting slightly slower causing a second minor peak. Only fractions of the later half of the major cathepsin protease peak and the most of the minor peak were collected in an attempt to obtain cathepsin proteases free of other proteins. These fractions were analyzed by non-reducing SDS-PAGE and revealed a dominant protein complex at 26 kD in all fractions. Fraction 1 contained other low molecular weight species. When those fractions containing only the 26 kD species were pooled and further characterized by Western blot and SDS-PAGE under reducing and non-reducing conditions, heterogeneity was apparent. Within the reduced preparation was a predominant couplet of 28kD and less intensely staining species of 14, 15, 17 and 20 kD whereas the non-reduced material was relatively homogenous with a major complex at 26 kD. Two dimensional SDS-PAGE analysis of the purified excretory/ secretory cathepsin proteases resolved into several components at 28 kD, with at least three closel migrating, dominant species and 4 minor species of higher pi (Fig 7). Lower molecular weight species (at 20 and 15 kD) were also detected but at the same pi as the 3 most prominent components suggesting they are either fragments of forms of the cathepsin proteases which are disulphide linked oligomers or products of auto-proteolysis of the intact polypeptide. Enzymatic characterisation cfF. hepatica cathepsin proteases Of the two fluorogenic substrates (N-CBZ Arg NMeC and N-CBZ Phe Arg NMeC) tested for sensitivity to the proteolytic action of the purified proteases, only N-CBZ Phe Arg NMeC proved susceptible and was used in all further enzymatic characterisation. There was no detectable activity on N-CBZ Arg NMeC even at high concentrations of the enzyme. In determining the pH optimum of the cathepsin proteases, activity was highly influenced by the buffer ion used. A pH maximum of 7.2 was achieved in 100 mM phosphate, and activity was detected over a broad pH range (pH 6.5-9.0; Fig. 4). An average 1^ of 46μM was derived by a Michaelis- Menten plot for this group of cathepsin proteases (lOOmM phosphate, pH 7.45). In the Tris buffer, and using half the enzyme concentration, a pH maximum was not determined, with activity increasing in alkaline conditions (Fig. 8). The inhibition assays revealed that the cathepsin proteases were highly sensitive to very low concentrations of the classic inhibitors of this class of proteases (Table 3), although IAA was least effective with only 74% inhibition at a lOOμM concentration. PMSF had a very slight inhibitory effect, whereas aprotinin and EDTA caused no decrease in activity. DTT was found to be an absolute requirement for activity (Table 3). Sequence homology to the thiol cathepsins An adult F. hepatica expression cDNA library was screened with the ovine anti- cathepsin protease serum. High levels of reactivity was observed and after extensively absorbing the antisera with E. coli, approximately 0.1-1% of the library was still positive. Nine randomly selected positive clones were purified and their cDNA inserts were partially sequenced. They were all found to be identical in sequence at their 3' end. The insert of the largest clone, Fhcatl was sequenced in both strands and on analysis revealed an open reading frame that extended from nucleotides 25 to 1002 (Fig. 9A). The predicted open reading frame encodes a putative protein of 326. amino acids in length which has homology to members of the thiol cathepsin family of cathepsin proteases (E.C. 3.4.22.-) (Fig. 10). The putative Fhcatl preproprotein has 44% homology to human preprocathepsin L and 39% homology with preprocathepsin H. There was limited homology to human preprocathepsin B (23%) and cathepsin proteases from Schistosoma (20%, Fig. 10) and Haemonchus (20%). The nucleotide and predicted amino acid sequences of another cDNA sequence located in the screen of the cDNA library, Fhcat2, is shown in Figure 9B. It is noted that Fhcat2 has 87% homology to Fhcatl. By homology to the mammalian thiol cathepsins, amino acids 1-17 of the putative Fhcatl preproprotein encode the Pre region and amino acids 18-107 encode the Pro region. The Pre and Pro regions are cleaved off to form a mature protease of 219 amino acids in length. Residues 126-136 of die predicted Fhcatl mature protein contains the thiol cathepsin consensus pattern Q-x(3)-[GE]-x-C-W-x(2)-[STAG] and subsequently the best homologies to the thiol cathepsins are found on comparison to the mature polypeptides, with 54% and 44% identity to cathepsins L and H (Fig. 10). Surprisingly, good homologies were obtained with various plant thiol cathepsins (bromelain: 39% (Fig. 10), barley aleurain: 47%, actinidin: 42%, papain: 35%). To ensure that the cloned cDNA was representative of the secreted/excreted cathepsin proteases of F. hepatica, purified peptides were generated from chrymotryptic and endo-Glu-C digests of the purified cathepsin proteases. One series of peptides (CT21.2, GC15.2 and CT13.3) overlapped with each other and their contiguous sequence aligns to positions 188-206 of the putative Fhcatl polypeptide (Fig. 9A). Peptide CT13.3 displayed one site of polymorphism at position 204 where both Tyr and Cys were detected. A second series of peptides (CT13.2 and CT11.3) were also contiguous when mapped to positions 277-291 of the predicted amino acid sequence of the Fhcatl cDNA (Fig. 9A). CT11.3, in particular has a highly conserved region containing the Asn residue which makes up part of the catalytic triad of the thiol cathepsins (Fig. 9A and Musil et al. 1991). N-terminal sequences cfF. hepatica cathepsin proteinases Direct N-terminal sequencing of the purified cathepsin proteases revealed a single major sequence which was representative of N-termini of other members of the thiol cathepsin family (Fig. 11). Endo-Glu-C and chymotryptic digests also yielded peptides (GC20.2, GC3.1) that could be aligned to die N-termini of thiol cathepsins (Fig. 11) and matched the predicted N-terminus of the Fhcatl polypeptide (Fig. 9A). However the peptide sequence and the direct N-terminal sequence were not identical. In both cases die highly conserved proline close to die N-terminus was present. Similarly, the DWR motif conserved in the cathepsin L and H subfamilies was intact in the two putative F. hepatica N-terminal sequences. Beyond these conservations the sequences differ in several respects. The endo-Glu-C peptide, (GC20.2), in agreement with the N-terminus of bromelain, has two residues prior to the conserved proline. This peptide terminated with a Glu at position 9 but it is likely to be contiguous with the peptide GC3.1 which aligns at this exact region with chicken cathepsin L (Fig. 11). This GC20.2 sequence has 78% identity with the bromelain N-terminus but also has similar levels of homology with cathepsins L and H N-termini. However, the direct N-terminal sequence has only the one residue (valine) prior to the proline in position two, a feature shared with most tiiiol cathepsins. This sequence then deviates from most of the thiol cathepsin N-terminal sequences after the DWR region resulting in a lower level of homology to bromelain (61%) and the cadiepsin L subfamily. HydraxyprcΛines in the excreted! secreted cathepsin proteinases c F. hepatica Amino acid sequencing of peptides GCI 5.2 and GC20.2 yielded an unusual product at cycles 4 and 3 respectively in the absence of any other phenylthiohydantion amino acid derivatives (PTH-amino acid). Small amounts of an identically eluting product was also detected in N-terminal sequencing runs of the purified preparations of the Fasciola cathepsins, along with significant amounts of PTH-proline. Since good homologies of these sequences to regions in other thiol cathepsins (Fig. 10 and 11) suggested a proline would be predicted at these sites, the possibility of a modified proline within these peptides was investigated. The most simple modification of proline occurs in the collagen chains where proline is hydroxylated at either C3 or C4 generating 3-hydroxyproline (3-Hyp) or 4- hydroxyproline (4-Hyp) respectively. To determine if the unusual PTH amino acid derived from d e Fasciola cathepsin proteases were indeed hydroxylated prolines, both 3-Hyp-PTH and 4-Hyp-PTH derived from the CB6 peptides of die α j and α chains of human type III collagen were chromatographically compared. Partial hydroxylation of proline to 3-Hyp and 4-Hyp has been demonstrated in the a→ collagen chain CB6 peptide which has the sequence: G P/3-Hyp P/4-Hyp G L A G P P P/4-Hyp. On Edman degradation of this peptide a small amount of 3-Hyp-PTH was detected at cycle 2 accompanied by large amounts of PTH-proline, whereas the 2 major adducts of 4-Hyp-PTH are distinctive in cycles 3 and 9. Similarly, we detected partial but more significant modification of die proline to 3-Hyp in the second cycle of sequencing the α CB6 peptide (G 3-Hyp/P R G A P G). When the chromatograms of the collagen derived 3-Hyp-PTH are aligned widi those of the unidentified Edman degradation product derived from the cathepsin protease peptides, co-elution was obvious. In both these purified Fasciola peptides, PTH- proline was also detected as a minor product, 16% and 12% of total proline. N- terminal sequencing of the purified Fasciola cathepsin proteases, however, indicated that hydroxylation near the N-terminus was a minor event with only 12 and 21% of prolines found in the hydroxylated form in 2 different sequencing runs. These data assumed d at both 3-Hyp and proline behave similarly on Edman degradation. Significantly, no 4-Hyp-PTH was detected in any amino acid sequencing of die Fasciola products. Controls Immunized 1 Animal Worm FEC FEC/worm Animal Worm FEC FEC/worm N Number Burden egg/g number Burden egg/g 391 42 1215 28.9 398 105 1 10 1.05 1 392 74 340 4.6 415 106 305 2.88 I 396 49 890 18.2 418 56 210 3.75 41 1 90 580 6.4 435 40 80 2.00 420 86 975 1 1.3 456 81 180 2.22 433 87 765 8.8 457 39 230 5.90 454 99 1265 12.8 459 138 205 1.48 466 34 320 9.4 475 146 350 2.09 468 82 250 3.0 479 43 265 6.16 469 53 400 7.5 480 39 125 3.20 Average 69.6 700 11.1 Average 79.3 206 3.10 S.D. 23.0 377 7.6 S.D. 42.1 86 1.73 c.v. 33.0 53.9 68.5 c.v. 53.0 41.9 55.8 %reduction 0 69.7 67.4 geo. mean 65.7 604 9.2 geo. mean 69.6 188 2.7 %reduction 5.3 69.4 68.0 Table I. Summary of worm burdens and final FECs and worm fecundity (FEC woim) of individual animals from control and immunized groups infected with F. hepatica metacercariae. (c.v. = coefficient of variation, geo. mean = geometric mean). Table 2. Percent egg viability of controls and cysteine protease vaccinates. ♦ Statistical data on the control group excludes those animals retuming eggs showing dormancy (i.e. 0% viability). Table 3 Inhibition of F. hepatica cysteine proteases by known protease inhibitors. EXAMPLE 3 EFFICACY OF RECOMBINANT VACCINE The recombinant vaccine trial was performed as described on page 26 except tha 120μg of protein was given per dose. Groups of ten sheep received either nativ denatured cathepsins (Group A), native active cathepsins (Group B), Fhcat2 (Grou C), Fhcatl (Group D) and Fhcatl +2 (Group E). Group G received PBS alone whilst Group E were the uninfected controls. 1. Purification of Native active and Native denatured cathepsin proteases fro Fasciola hepatica Adult flukes were isolated from experimentally infected sheep and washed in PBS (3 x 10 minutes) and then were incubated in RPMI at 37 β C for 2 hours. This extrac was frozen at 20 °C and when needed thawed at 4 °C. The extract was centrifuge at 10,000 xg to remove eggs and particulate material before filtering on a 0.45 micron filter. A Minitan concentrator (Millipore) with a 10,000 Dalton molecular weight cutoff membrane was then used to reduce the volume of the filtrate from approximately 2 litres to approximately 100 mis. This solution was further concentrated and simultaneously dialysed against 50 mM Tris pH 7.5, IM NaCl (Buffer 1), to 1 ml using a microprodicon concentrator using a 10,000 Dalton molecular weight cutoff membrane. Gel filtration chromatography was carried out using Buffer 1 and a Pharmacia FPLC with a Superose 12 column at a flow rate o 0.25 ml/minute and 1 ml fractions collected. These fractions were analysed usin SDS-PAGE and Western blotting on reducing and non-reducing gels, as well zymogram analysis. Fractions that showed homogeneity by these criteria were use for preparing the antigen for vaccination. Antigen that was to be given to Group was incubated in 1% w/v SDS for 30 minutes and then acetone precipitated an resuspended in PBS. 2. Production of clones expressing recombinant Fhcatl and Fhcat2 DNA encoding the mature cathepsin was amplified from Fhcatl and Fhcat2 containing plasmids using the following primers: ICEF 5 ' ACAGCTCGAGGATCCGGCTGTACCCGACA 3' (SEQ ID NO. 19); ICER 5' CTCGAGGATCCTATCACGGAAATCGTGC 3' (SEQ ID NO. 20). The PCR products were cut with BamHI and then ligated into the BamHI site of pET15b (Novagen, USA). The ligated plasmid DNA was transformed into the E. coli strain BL21 (DE3)pLysS. Following transformation the cells were plated on L Broth agar plates containing ampicillin (20 μg/ml) and chloramphenicol (25 μg/ml). Recombinant clones were screened for their ability to express Fhcat by the following manner. Individual colonies were picked and used to inoculate one ml of L-Broth containing ampicillin (20 μg/ml), chloramphenicol (25 μg/ml) and 1 mM IPTG and grown at 37 °C for 3 hours after which the cells were collected by centrifugation. The cell pellets were then resuspended in sample buffer (Laemmli, 1970) and heated to 95 °C for 5 minutes before being loaded onto 15% w/v polyacrylamide gels. After electrophoresis the gels were stained in Coomassie blue. Clones expressing an abundant protein of approximately 30 kDa were selected. Western blotting using a sheep anti-cathepsin antisera confirmed that the selected clones expressed recombinant Fhcat. Purification of recombinant Fhcat protein Clones encoding the mature form of Fhcatl or 2 were grown overnight in 10 mis of L-broth containing ampicillin (20 μg/ml) and chloramphenicol (25 μg/ml). They were then inoculated into 200 mis of L-Broth containing ampicillin (20μg/ml), ,chloramphenicol (25 μg/ml) and 1 mM IPTG and grown at 37 °C for 3 hours. The cells were collected by centrifugation (10,000g for ten minutes) and then the cell pellets were resuspended in 50 mis of PBS, 0.1% v/v Triton X-100. Following a freeze /thaw the cells were lysed and the insoluble material collected by centrifugation (10,000g for ten minutes). The insoluble material was then resuspended in 10 mis of 8 M urea in lx binding buffer and heated to 65 °C for ten minutes. The insoluble material was removed by centrifugation and the supernatant was kept. Recombinant protein was purified from the supernatant by chromatography using His. Bind. Resin as described by the manufacturer (Novagen, USA) using denaturing conditions containing 8M urea. The eluted material (approximately 10 mis) was dialysed against 2 changes of one litre of PBS at 4 °C. During dialysis the recombinant proteins precipitate out of solution. The dialysed solutions were then sonicated to form a fine suspension and the protein concentration was determined. 3. Results Fecal egg count data were obtained in sheep at week 14 and 15 post infection. The egg counts are listed by group (10 sheep per group) with the mean ± SD and percentage reduction and p value. The Groups in the trial were as follows: GROUP VACCINE A Native denatured adult cathepsin protease (ACP) B Native active ACP C Fhcat 2 D Fhcat 1 E Fhcat 1 + 2 F Infected controls G Uninfected controls The data show that vaccination of sheep with the native active cathepsin proteases induces a 67-75% reduction in fecal egg counts (FEC) at weeks 14-15 post infection. This result is significant (p < .01). Vaccination with denatured cathepsin protease or Fhcat 2 also induces a significant reduction in FEC at week 14 (59%) (P < 0.013). Fhcat 2 also induces a significant 51% reduction in FEC at week 15 (p < .036). Thus, vaccination with the recombinant Fhcat 2 protein mimics the efficacy of the native cathepsin protease from Fhepatica. TABLE 4 Week 14 VACCINE GROUP 1 A B C D E F G Average 73.7 59.9 74.9 127.5 164.6 183.6 0 StDev 68.6 85.7 61.8 112.9 82.5 107.5 0 % reduction 59.8584 67.3747 59.20479 30.5556 10.3486 0 0 P = value 0.013 0.01 0.012 0.27 ND 2 1 Fecal egg count data (eggs per gram of feces) 2 ND, not determined; P>0.05 TABLE 5 Week 15 VACCINE GROUP 1 - A B C D E F G Average 114 52.2 103.9 200 213.7 216 0 StDev 124 59 66 150 98 146 0 % reduction 47.????2 75.8333 51.898148 7.40741 1.0648 0 0 P = value 0.107 0.003 0.036 ND 2 ND 2 1 Fecal egg count data (eggs per gram of feces) ~ ND, not determined; P>0.05 Summary of fecal egg counts from the recombinant vaccine trial at weeks 14 and 15 post infection from animals vaccinated as shown on page 39. EXAMPLE 4 SEQUENCE ANALYSIS OF F. HEPATICA LARVAL CATHEPSIN PROTEASES Larval proteases were isolated and the N-terminal sequence determined and compared to cathepsin B of bovine, rat, mouse and human origin; cathepsin sequences of mouse and human origin; Fhcatl and cathepsin L sequences in a peptide GC20.2 from F. hepatica; schistosome cathepsin B sequences; and the plant proteases bromelain and papain. The results are set forth below: x Larval protease #1 L P E S F D A R Q 2 Larval protease #2 V P A S F D A R Q Bovine Cat B L P E s F D A R E 8 Rat Cat B L P E s F D A R E 8 Mouse Cat B L P E T F D A R E 7 Human Cat B L P A s F D A R E 7 Mouse Cat L I P K s V D W R E 4 Human Cat L A P R s V D W R E 4 Adult Fluke: N-Term 1 V P E D I D W R G 4 Peptide GC20.2 A V P D K I D W R E 3 Fhcatl A V P D K I D W R E 3 Schistosoma mansoni CAT B E I P S N F D s R K 4 Schistosoma japonicum CAT B E I P S Q F D S R K 4 Bromelain A V P Q s I D W R D 4 Papain I P E Y V D W R Q 5 1 SEQ ID NO. 21 ; 2 SEQ ID NO . 22 The numbers on the right indicate the number of residues in each sequence that are identical with the sequence of larval protease # 1. EXAMPLE 5 CLONING OF CATHEPSIN B PROTEASE FROM LARVAL F.HEPATICA Larva of F. epatica were excysted (Carmona et al.,1993) in vitro and total RNA extracted using Ultraspec as recommended by the manufacturer (Biotecx Laboratories, Houston, Texas). cDNA first strand synthesis was performed using M- MLV reverse transcriptase primed with oligo dT (Sambrook et al.,1989). Amplification of the cathepsin B cDNA was performed using an oligonucleotide predicted from the N-terminal amino acid sequence shown in Example 4 (SEQ ID NO. 21) and oligo dT. The amplified Cathepsin B cDNA was cloned using the pCR- Script SK( + ) Cloning Kit (Stratagene, La Jolla, USA). One clone (FhcatBl) was obtained. Plasmid DNA was isolated and DNA sequence determined as described on page 30 above. DNA translation and alignments of the predicted amino acid sequence were carried out using Staden software and sequences from Genbank. As shown in Figure 12, the FhcatBl sequence predicts a polypeptide showing high similarity to the cathepsin B family of proteases. In particular, cathepsin B sequences from human and S. mansoni are, similar to Fhcat BI. FhcatBl shows a lower degree of similarity to cathepsin L sequences from human and F. hepatica. Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. REFERENCES BARRETT, A.J. 1980. Fluorimetric assays for cathepsin B and cathepsin H with methylcourylamide substrates. Biochemical Journal 187:909-912. BIRNBOIM, H.C. AND DOLY, J. 1979 Nucleic Acids Research, 7, 1513-1518. BURNETTE, W.N. 1981. 'Western Blotting': electrophoretic transfer of proteins from sodium dodecyl sulphate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analytical Biochemistry 112:195-203 CARMONA, C, DOWD, A.J., SMITH, A.M. AND DALTON, J.P. 1993. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol. Biochem. Parasitόl. 62:9-18. CHAN, S.-J., SEGUNDO, B.S., MCCORMICK, M.B. & STEINER, D.F. 1986. Nucleotide and predicted amino acid sequences of cloned human and house preprocathepsin B cDNAs. Proceedings of the National Academy of Science 83:7721-7725. COLES, G.C. & RUBANO, D. 1988. Antigenicity of a proteolytic enzyme of Fasciola hepatica Journal of Helminthology 62:257-260. DALTON, J.P. & HEFFERNAN, M. 1989. Thiol proteases released in vitro by Fasciola hepatica Molecular and Biochemical Parasitology 35: 161 -166. DRENTH, J., JANSONIUS, J.N., KOEKSEK, R. & WOLTHERS, B.G.1971. The structure of papain. Advances in Proetin Chemistry 25:79-115 EAKIN, A.E., MILLS, A.A., HARTH, G., MCKERROW, J.H. & CRAIK, C.S. 1991. The sequence, organisation, and expression of the major protease (Cruzipain) from Trypanosoma cruzi. Journal of Biological Chemistry 267: '411-7240. HAROUN, E.M. AND HILLYER, G.V. 1986. Vet. Parasitol 2Q.-63. JOSEPH, L.J., CHANG, L.C., STAMENKOVICH, D AND SUKHATME, V.P. 1988. Complete nucleotide and deduced amino acid sequences of human and murine preprocathepsin L. J. din. Invest. 81: 1621-1629. LAEMMLL U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. MASON, R.W., WALKER, J.E. & NORTHROP, F.D. 1986. The N-terminal amino acid sequences of the heavy and light chains of human cathepsin. L. Relationship to a cDNA clone for a major cathepsin proteinase from a mouse macrophage cell line. Biochemical Journal 240:373-377. Mc MANUS, D., WAINE, G., WEN, Y., BECKER, M., KALBMNA, B., LIU, X., AND TIU, W. 1993. Towards a vaccine against Asian schistosomiasis. Today's Life Science MORRISSEY, J. H. 1981. Silver stain for proteins in polyacrylamide gels: A modified procedure with enhanced uniform sensitivity. .Analytical Biochemistry 117:307-310. MOTTRAM, CM., NORTH, M.J., BARRY, J.D. & COOMBS, G.H. 1989. A cathepsin proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C- terminal extension. FEBS Letters 258:211-215. MUSIL, D., ZUCIC, D., TURK, D., ENGH, R.A., MAYR, I., HUBER, R., POPOVIC, T., TURK, V., TOWATARI, T., KATUNUMA, N. & BODE, W. 1991. The refined 2.15A X- ray crystal structure of human liver cathepsin G: the structural basis for its specificity. European Molecular Biology Organisation Journal 10:2321-2330. O'FARRELL, P. H. 1975. High resolution two dimensional electrophoresis of proteins. Journal Biological Chemistry 250, 4007-4021. RΠΌNJA, A., ROWAN, A.D., BUTTLE, D.J., RAWLINGS, ND., TURK, V. & BARRETT, A.J. 1989. Stem bromelain: Amino acid sequence and implications of weak binding of cystatin. Federation of European Biochemical Societies Letters 247:419-424. SAMBROOK, J., et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Sprin Harbor, NY, USA. SANGER, F., NICKLEN, S. AND COULSON, A.R. (1977) Proceedings of the National Academy of Science USA, 74, 5463-5469. SEXTON, J.L., MILNER, A.R., PANACCIO, M., WIJFFELS, G., CHANDLER, D., THOMPSON, C, WILSON, L., SPΠΉILL, T.W., MITCHELL, G.F. & CAMPBELL, NJ. 1990. Glutathione S-transferase:Novel vaccine against Fasciola hepatica infection in sheep. Journal of Immunology 145:3905-3910. SONODA, S. & SCHALAMOWΓΓZ, M. 1970. Studies of I 125 trace labelling of immunoglobulin G by Chloramine T. Immunochemistry Vol. 7: 885-898. SPITHILI-, T.W. 1992. Control of tissue parasites. 3. Trematodes in Animal parasite control utilizing biotechnology, ed. W.K. Yong, CRC press, Boca Raton. VAN KAMPEN, E J. & ZULSTRA, W.G. 1961. Standardization of hemoglobinometry. II The hemiglobincyanide method. Clinica Chimica Acta 6:538 WIEDERANDERS, B., BROEMME, D., KIRSCHKE, H., KALKKINEN, N., RINNE, A., PAQUETTE, T. & TOOTHMAN, P. 1991. Primary structure of bovine cathepsin S. Comparison to cathepsins L, H, B and papain. Federation of European Biochemical Societies Letters 286:189-192. WIJFFELS, G.L., SEXTON, J.L., SALVATORE, L., PETTΠT, J.M., HUMPHRIES, D.C., PANACCIO, M. & SPΠΉΠLL, T.W. 1992. Experimental Parasitology 74:8 '-99. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: DARATECH PROPRIETARY LIMITED INVENTORS: MILNER, PANACCIO, SPITHILL and WIJFFELS (ii) TITLE OF INVENTION: A VACCINE AND POLYPEPTIDES USEFUL FOR SAME (iii) NUMBER OF SEQUENCES: 24 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: DAVIES COLLISON CAVE (B) STREET: 1 LITTLE COLLINS STREET (C) CITY: MELBOURNE (D) STATE: VICTORIA (E) COUNTRY: AUSTRALIA (F) ZIP: 3000 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT - INTERNATIONAL (B) FILING DATE: 04-FEB-1994 (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: AU PL7109 (B) FILING DATE: 05-FEB-1993 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: HUGHES, E JOHN L (C) REFERENCE/DOCKET NUMBER: EJH/EK (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (613) 254 2777 (B) TELEFAX: (613) 254 2770 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1075 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 25..1002 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGAACGTCCG ATAATAATCC AAAA ATG CGA TTG TTC ATA TTA GCC GTC CTC 51 Met Arg Leu Phe He Leu Ala Val Leu 1 5 ACA GTC GGA GTG CTT GGC TCG AAT GAT GAT TTG TGG CAT CAG TGG AAG 99 Thr Val Gly Val Leu Gly Ser Asn Asp Asp Leu Trp His Gin Trp Lys 10 15 20 25 CGA ATG TAC AAT AAA GAA TAC AAT GGG GCT GAC GAT CAG CAC AGA CGA 147 Arg Met Tyr Asn Lys Glu Tyr Asn Gly Ala Asp Asp Gin His Arg Arg 30 35 40 AAT ATT TGG GAA AAG AAT GTG AAA CAT ATC CAA GAA CAT AAC CTA CGT 195 Asn He Trp Glu Lys Asn Val Lys His He Gin Glu His Asn Leu Arg 45 50 55 CAC GAT CTC GGC CTC GTC ACC TAC ACA TTG GGA TTG AAC CAA TTC ACG 243 His Asp Leu Gly Leu Val Thr Tyr Thr Leu Gly Leu Asn Gin Phe Thr 60 65 70 GAT ATG ACA TTC GAG GAA TTC AAG GCC AAA TAT CTA ACA GAA ATG TCA 291 Asp Met Thr Phe Glu Glu Phe Lys Ala Lys Tyr Leu Thr Glu Met Ser 75 80 85 CGC GCG TCC GAT ATA CTC TCA CAC GGT GTC CCG TAT GAG GCG AAC AAT 339 Arg Ala Ser Asp He Leu Ser His Gly Val Pro Tyr Glu Ala Asn Asn 90 95 100 105 CGT GCC GTA CCC GAC AAA ATT GAC TGG CGT GAA TCT GGT TAT GTG ACG 387 Arg Ala Val Pro Asp Lys He Asp Trp Arg Glu Ser Gly Tyr Val Thr 110 115 120 GAG GTG AAA GAT CAG GGA AAC TGT GGT TCC TGT TGG GCA TTC TCA ACA 435 Glu Val Lys Asp Gin Gly Asn Cys Gly Ser Cys Trp Ala Phe Ser Thr 125 130 135 ACC GGT ACT ATG GAG GGA CAG TAT ATG AAA AAC GAA AGA ACT AGT ATT 483 Thr Gly Thr Met Glu Gly Gin Tyr Met Lys Asn Glu Arg Thr Ser He 140 145 150 TCA TTC TCT GAG CAA CAA CTG GTC GAT TGT AGT GGT CCT TGG GGA AAT 531 Ser Phe Ser Glu Gin Gin Leu Val Asp Cys Ser Gly Pro Trp Gly Asn 155 160 165 AAT GGT TGC AGT GGT GGA TTG ATG GAA AAT GCT TAC CAA TAT TTG AAA 579 Asn Gly Cys Ser Gly Gly Leu Met Glu Asn Ala Tyr Gin Tyr Leu Lys 170 175 180 185 CAA TTT GGA TTG GAA.ACC GAA TCC TCT TAT CCG TAC ACG GCT GTG GAA 62 Gin Phe Gly Leu Glu Thr Glu Ser Ser Tyr Pro Tyr Thr Ala Val Glu 190 195 200 GGT CAG TGT CGA TAC AAT AAG CAG TTA GGA GTT GCC AAA GTG ACT GGC 67 Gly Gin Cys Arg Tyr Asn Lys Gin Leu Gly Val Ala Lys Val Thr Gly 205 210 215 TAC TAC ACT GTG CAT TCT GGC AGT GAG GTA GAA TTG AAA AAT CTA GTC 72 Tyr Tyr Thr Val His Ser Gly Ser Glu Val Glu Leu Lys Asn Leu Val 220 225 230 GGA GCC CGA AGA CCT GCC GCG GTC GCT GTG GAT GTG GAA TCT GAC TTC 77 Gly Ala Arg Arg Pro Ala Ala Val Ala Val Asp Val Glu Ser Asp Phe 235 240 245 ATG ATG TAC AGG AGT GGT ATT TAT CAG AGC CAA ACT TGT TCA CCG CTT 81 Met Met Tyr Arg Ser Gly He Tyr Gin Ser Gin Thr Cys Ser Pro Leu 250 255 260 265 CGT GTG AAT CAT GCA GTC TTG GCT GTC GGT TAC GGA ACA CAG GGT GGT 86 Arg Val Asn His Ala Val Leu Ala Val Gly Tyr Gly Thr Gin Gly Gly 270 275 280 ACT GAC TAT TGG ATT GTG AAA AAT AGT TGG GGA ACG TAC TGG GGT GAG 91 Thr Asp Tyr Trp He Val Lys Asn Ser Trp Gly Thr Tyr Trp Gly Glu ■ 285 290 295 CGC GGT TAC ATT CGA ATG GCT AGG AAT CGA GGT AAC ATG TGT GGA ATT 96 Arg Gly Tyr He Arg Met Ala Arg Asn Arg Gly Asn Met Cys Gly He 300 305 310 GCT TCG CTG GCC AGT CTC CCG ATG GTG GCA CGA TTT CCG TGATACGTTT 101 Ala Ser Leu Ala Ser Leu Pro Met Val Ala Arg Phe Pro 315 320 325 CTGTTATTAT GAAAACGCAC CAAACAATTA ATTTCATTCA GCTTTGCTTC AAAAAAAAAA 107 AAA 107 (2) INFORMATION FOR SEQ ID NO:2: (i) SE Q UENCE CHARACTERISTICS: (A) LENGTH: 326 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Arg Leu Phe He Leu Ala Val Leu Thr Val Gly Val Leu Gly Ser 1 5 10 15 Asn Asp Asp Leu Trp His Gin Trp Lys Arg Met Tyr Asn Lys Glu Tyr 20 25 30 Asn Gly Ala Asp Asp Gin His Arg Arg Asn He Trp Glu Lys Asn Val 35 40 45 Lys His He Gin Glu His Asn Leu Arg His Asp Leu Gly Leu Val Thr 50 55 60 Tyr Thr Leu Gly Leu Asn Gin Phe Thr Asp Met Thr Phe Glu Glu Phe 65 70 75 80 Lys Ala Lys Tyr Leu Thr Glu Met Ser Arg Ala Ser Asp He Leu Ser 85 ' 90 95 His Gly Val Pro Tyr Glu Ala Asn Asn Arg Ala Val Pro Asp Lys He 100 105 110 Asp Trp Arg Glu Ser Gly Tyr Val Thr Glu Val Lys Asp Gin Gly Asn 115 120 125 Cys Gly Ser Cys Trp Ala Phe Ser Thr Thr Gly Thr Met Glu Gly Gin 130 135 140 Tyr Met Lys Asn Glu Arg Thr Ser He Ser Phe Ser Glu Gin Gin Leu 145 150 155 160 Val Asp Cys Ser Gly Pro Trp Gly Asn Asn Gly Cys Ser Gly Gly Leu 165 170 175 Met Glu Asn Ala Tyr Gin Tyr Leu Lys Gin Phe Gly Leu Glu Thr Glu 180 185 190 Ser Ser Tyr Pro Tyr Thr Ala Val Glu Gly Gin Cys Arg Tyr Asn Lys 195 200 205 Gin Leu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr Val His Ser Gly 210 215 220 Ser Glu Val Glu Leu Lys Asn Leu Val Gly Ala Arg Arg Pro Ala Ala 225 230 235 240 Val Ala Val Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser Gly He 245 250 255 Tyr Gin Ser Gin Thr Cys Ser Pro Leu Arg Val Asn His Ala Val Leu . 260 265 270 Ala Val Gly Tyr Gly Thr Gin Gly Gly Thr Asp Tyr Trp He Val Lys 275 280 285 Asn Ser Trp Gly Thr Tyr Trp Gly Glu Arg Gly Tyr He Arg Met Ala 290 295 300 Arg Asn Arg Gly Asn Met Cys Gly He Ala Ser Leu Ala Ser Leu Pro 305 310 315 320 Met Val Ala Arg Phe Pro 325 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Val Pro Glu Asp He Asp Trp Arg Gly Tyr Tyr Tyr Val 1 5 10 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ala Val Pro Asp Lys He Asp Trp Arg Glu 1 5 10 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Ser Gly Tyr Val Thr Glu 1 5 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gly Leu Glu Thr Glu Ser Ser Tyr 1 5 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ser Ser Tyr Pro Tyr Thr Ala Val Glu 1 5 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid ' (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Thr Ala Val Glu Gly Gin Cys Arg Tyr 1 5 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Gly Thr Gin Gly Gly Thr Asp Tyr Trp 1 5 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 6 amino acids .(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: He Val Lys Asn Ser Trp 1 5 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1041 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 8..985 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GCAAACA ATG AGA TTG GTA ATC CTA ACC CTA CTC ATC GTC GGA GTG TTC 49 Met Arg Leu Val He Leu Thr Leu Leu He Val Gly Val Phe 1 5 10 GCC TCA AAT GAC GAT TTG TGG CAT CAA TGG AAG CGA ATT TAC AAT AAA 97 Ala Ser Asn Asp Asp Leu Trp His Gin Trp Lys Arg He Tyr Asn Lys 15 20 25 30 GAA TAC AAG GGA GCT GAC GAT GAC CAC AGG AGA AAT ATT TGG GAA CAA 145 Glu Tyr Lys Gly Ala Asp Asp Asp His Arg Arg Asn He Trp Glu Gin 35 40 45 AAT GTG AAA CAT ATC CAA GAA CAC AAC CTG CGC CAC GAT CTC GGT CTC 193 Asn Val Lys His He Gin Glu His Asn Leu Arg His Asp Leu Gly Leu 50 55 60 GTC ACC TAC AAG TTG GGA TTG AAC CAA TTC ACC GAT ATG ACA TTC GAG 241 Val Thr Tyr Lys Leu Gly Leu Asn Gin Phe Thr Asp Met Thr Phe Glu 65 70 75 GAA TTC AAA GCC AAA TAT CTA ACA GAA ATG CCA CGC GCG TCT GAG TTA 289 Glu Phe Lys Ala Lys Tyr Leu Thr Glu Met Pro Arg Ala Ser Glu Leu 80 85 90 CTC TCA CAC GGT ATC CCA TAT AAG GCT AAC AAG CGT GCT GTA CCC GAC 337 Leu Ser His Gly He Pro Tyr Lys Ala Asn Lys Arg Ala Val Pro Asp 95 100 105 110 AGA ATT GAC TGG CGT GAA TCC GGT TAT GTG ACG GAG GTG AAA GAT CAG 385 Arg He Asp Trp Arg Glu Ser Gly Tyr Val Thr Glu Val Lys Asp Gin 115 120 125 GGA GGC TGT GGT TCT TGT TGG GCT TTC TCA ACA ACA GGT GCT ATG GAA 43 Gly Gly Cys Gly Ser ' Cys Trp Ala Phe Ser Thr Thr Gly Ala Met Glu 130 135 140 GGA CAG TAT ATG AAA AAC GAA AAA ACT AGT ATT TCA TTC TCT GAG CAA 48 Gly Gin Tyr Met Lys Asn Glu Lys Thr Ser He Ser Phe Ser Glu Gin 145 150 155 CAA CTG GTC GAT TGT AGC GGT CCT TTT GGC AAT TAT GGT TGT AAT GGT 52 Gin Leu Val Asp Cys Ser Gly Pro Phe Gly Asn Tyr Gly Cys Asn Gly 160 165 170 GGA CTA ATG GAA AAT GCA TAC GAA TAT TTG AAA CGA TTT GGA TTG GAA 57 Gly Leu Met Glu Asn Ala Tyr Glu Tyr Leu Lys Arg Phe Gly Leu Glu 175 180 185 190 ACC GAG TCT TCT TAT CCT TAC AGG GCT GTG GAA GGA CAG TGT CGA TAC 62 Thr Glu Ser Ser Tyr Pro Tyr Arg Ala Val Glu Gly Gin Cys Arg Tyr 195 200 205 AAC GAG CAG TTG GGA GTT GCC AAA GTG ACT GGC TAC TAT ACG GTA CAT 67 Asn Glu Gin Leu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr Val His 210 215 220 TCT GGC GAT GAG GTA GAA TTG CAA AAT CTA GTC GGT TGC CGA AGA CCT 72 Ser Gly Asp Glu Val Glu Leu Gin Asn Leu Val Gly Cys Arg Arg Pro 225 230 235 GCT GCG GTC GCT TTG GAT GTG GAG TCA GAC TTC ATG ATG TAC AGG AGT 76 Ala Ala Val Ala Leu Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser 240 245 250 GGT ATT TAT CAG AGC CAA ACT TGT TCA CCG GAT CGT TTG AAC CAT GGA 81 Gly He Tyr Gin Ser Gin Thr Cys Ser Pro Asp Arg Leu Asn His Gly 255 260 265 270 GTG TTG GCT GTC GGT TAT GGA ATA CAG GAT GGT ACT GAC TAC TGG ATT 86 Val Leu Ala Val Gly Tyr Gly He Gin Asp Gly Thr Asp Tyr Trp He 275 280 285 GTG AAA AAC AGT TGG GGA ACG TGG TGG GGT GAG GAC GGT TAC ATT CGA 91 Val Lys Asn Ser Trp Gly Thr Trp Trp Gly Glu Asp Gly Tyr He Arg 290 295 300 ATG GTT AGG AAA AGA GGT AAC ATG TGT GGA ATT GCT TCT CTG GCC AGT 96 Met Val Arg Lys Arg Gly Asn Met Cys Gly He Ala Ser Leu Ala Ser 305 310 315 GTC CCG ATG GTG GCA CAA TTT CCG TGATACTTTT CTGTTATTAC GAAAACACAC 101 Val Pro Met Val Ala Gin Phe Pro 320 325 TGAATAATAA ATTTCACTCG GGAAAA 104 (2) INFORMATION FOR ' SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: Met Arg Leu Val He Leu Thr Leu Leu He Val Gly Val Phe Ala Ser 1 5 10 15 Asn Asp Asp Leu Trp His Gin Trp Lys Arg He Tyr Asn Lys Glu Tyr 20 25 30 Lys Gly Ala Asp Asp Asp His Arg Arg Asn He Trp Glu Gin Asn Val 35 40 45 Lys His He Gin Glu His Asn Leu Arg His Asp Leu Gly Leu Val Thr 50 55 60 Tyr Lys Leu Gly Leu Asn Gin Phe Thr Asp Met Thr Phe Glu Glu Phe 65 70 75 80 Lys Ala Lys Tyr Leu Thr Glu Met Pro Arg Ala Ser Glu Leu Leu Ser 85 90 95 His Gly He Pro Tyr Lys Ala Asn Lys Arg Ala Val Pro Asp Arg He 100 105 110 Asp Trp Arg Glu Ser Gly Tyr Val Thr Glu Val Lys Asp Gin Gly Gly 115 120 125 Cys Gly Ser Cys Trp Ala Phe Ser Thr Thr Gly Ala Met Glu Gly Gin 130 135 140 Tyr Met Lys Asn Glu Lys Thr Ser He Ser Phe Ser Glu Gin Gin Leu 145 150 155 160 Val Asp Cys Ser Gly Pro Phe Gly Asn Tyr Gly Cys Asn Gly Gly Leu 165 170 175 Met Glu Asn Ala Tyr Glu Tyr Leu Lys Arg Phe Gly Leu Glu Thr Glu 180 185 190 Ser Ser Tyr Pro Tyr Arg Ala Val Glu Gly Gin Cys Arg Tyr Asn Glu 195 200 205 Gin Leu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr Val His Ser Gly 210 215 220 Asp Glu Val Glu Leu Gin Asn Leu Val Gly Cys Arg Arg Pro Ala Ala 225 230 235 240 Val Ala Leu Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser Gly He 245 250 255 Tyr Gin Ser Gin Thr Cys Ser Pro Asp Arg Leu Asn His Gly Val Leu 260 265 270 Ala Val Gly Tyr Gly He Gin Asp Gly Thr Asp Tyr Trp He Val Lys 275 280 285 Asn Ser Trp Gly Thr Trp Trp Gly Glu Asp Gly Tyr He Arg Met Val 290 295 300 Arg Lys Arg Gly Asn Met Cys Gly He Ala Ser Leu Ala Ser Val Pro 305 310 315 320 Met Val Ala Gin Phe Pro 325 (2) INFORMATION FOR SEQ ID NO:13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: Met Arg Leu Phe He Leu Ala Val Leu Thr Val Gly Val Leu Gly Ser 1 5 10 15 Asn Asp Asp Leu Trp His Gin Trp Lys Arg Met Tyr Asn Lys Glu Tyr 20 25 30 Asn Gly Ala Asp Asp Gin His Arg Arg Asn He Trp Glu Lys Asn Val 35 40 45 Lys His He Gin Glu His Asn Leu Pro His Asp Leu Gly Leu Val Thr 50 55 60 Tyr Thr Leu Gly Leu Asn Gin Phe Thr Asp Met Thr Phe Glu Glu Phe 65 70 75 80 Lys Ala Lys Tyr Leu Thr Glu Met Ser Arg Ala Ser Asp He Leu Ser 85 90 95 His Gly Val Pro Tyr Glu Ala Asn Asn Arg Ala Val Pro Asp Lys He 100 105 110 Asp Trp Arg Glu Ser Gly Tyr Val Thr Glu Val Lys Asp Gin Gly Asn 115 120 125 Cys Gly Ser Cys Trp Ala Phe Ser Thr Thr Gly Thr Met Glu Gly Gin 130 135 140 Tyr Met Lys Asn Glu Arg Thr Ser He Ser Phe Ser Glu Gin Gin Leu 145 150 155 160 Val Asp Cys Ser Gly Pro Trp Gly Asn Asn Gly Cys Ser Gly Gly Leu 165 170 175 Met Glu Asn Ala Tyr Gin Tyr Leu Lys Gin Phe Gly Leu Glu Thr Glu 180 185 190 Ser Ser Tyr Pro Tyr Thr Ala Val Glu Gly Gin Cys Arg Tyr Asn Lys 195 200 205 Gin Leu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr Val His Ser Gly 210 215 220 Ser Glu Val Glu Leu Lys Asn Leu Val Gly Ala Arg Arg Pro Ala Ala 225 230 235 240 Val Ala Val Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser Gly He 245 250 255 Tyr Gin Ser Gin Thr Cys Ser Pro Leu Arg Val Asn His Ala Val Leu 260 265 270 Ala Val Gly Tyr Gly Thr Gin Gly Gly Thr Asp Tyr Trp He Val Lys 275 280 285 Asn S.er Trp Gly Thr Tyr Trp Gly Glu Arg Gly Tyr He Arg Met Ala 290 295 300 Arg Asn Arg Gly Asn Met Cys Gly He Ala Ser Leu Ala Ser Leu Pro 305 310 315 320 Met Val Ala Arg Phe Pro 325 (2) INFORMATION FOR SEQ ID NO:14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 333 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: Met Asn Pro Thr Leu He Leu Ala Ala Phe Cys Leu Gly He Ala Ser 1 5 10 15 Ala Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gin Trp Thr Lys Trp 20 25 30 Lys Ala Met His Asn Arg Leu Tyr Gly Met Asn Glu Glu Gly Trp Arg 35 40 45 Arg Ala Val Trp Glu Lys Asn Met Lys Met He Glu Leu His Asn Gin 50 55 60 Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe 65 70 75 80 Gly Asp Met Thr Ser Glu Glu Phe Arg Gin Val Met Asn Gly Phe Gin 85 90 95 Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gin Glu Pro Leu Phe Tyr 100 105 110 Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro 115 120 125 Val Lys Asn Gin Gly Gin Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr 130 135 140 Gly Ala Leu Glu Gly Gin Met Phe Arg Lys Thr Gly Arg Leu He Ser 145 150 155 160 Leu Ser Glu Gin Asn Leu Val Asp Cys Ser Gly Pro Gin Gly Asn Glu 165 170 175 Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Phe Gin Tyr Val Gin Asp 180 185 190 Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pro Tyr Glu Ala Thr Glu 195 200 205 Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly 210 215 220 Phe Val Asp He Pro Lys Gin Glu Lys Ala Leu Met Lys Ala Val Ala 225 230 235 240 Thr Val Gly Pro He Ser Val Ala He Asp Ala Gly His Glu Ser Phe 245 250 255 Leu Phe Tyr Lys Glu Gly He Tyr Phe Glu Pro Asp Cys Ser Ser Glu 260 265 270 Asp Met Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Ser Thr 275 280 285 Glu Ser Asp Asn Asn Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly Glu 290 295 300 Glu Trp Gly Met Gly Gly Tyr Val Lys Met Ala Lys Asp Arg Arg Asn 305 310 315 320 His Cys Gly He Ala Ser Ala Ala Ser Tyr Pro Thr Val 325 330 (2) INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 212 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: Ala Val Pro Gin Ser He Asp Trp Arg Asp Tyr Gly Ala Val Thr Ser 1 5 10 15 Val Lys Asn Gin Asn Pro Cys Gly Ala Cys Trp Ala Phe Ala Ala He 20 25 30 Ala Thr Val Glu Ser He Tyr Lys He Lys Lys Gly He Leu Glu Pro 35 40 45 Leu Ser Glu Gin Gin Val Leu Asp Cys Ala Lys Gly Tyr Gly Cys Lys 50 55 60 Gly Gly Trp Glu Phe Arg Ala Phe Glu Phe He He Ser Asn Lys Gly 65 70 75 80 Val Ala Ser Gly Ala He Tyr Pro Tyr Lys Ala Ala Lys Gly Thr Cys 85 90 95 Lys Thr Asp Gly Val Pro Asn Ser Ala Tyr He Thr Gly Tyr Ala Arg 100 105 110 Val Pro Arg Asn Asn Glu Ser Ser Met Met Tyr Ala Val Ser Lys Gin 115 120 125 Pro He Thr Val Ala Val Asp Ala Asn Ala Asn Phe Gin Tyr Tyr Lys 130 135 140 Ser Gly Val Phe Asn Gly Pro Cys Gly Thr Ser Leu Asn His Ala Val 145 150 155 160 Thr Ala He Gly Tyr Gly Gin Asp Ser He He Tyr Pro Lys Lys Trp 165 170 175 Gly Ala Lys Trp Gly Glu Ala Gly Tyr He Arg Met Ala Arg Asp Val 180 185 190 Ser Ser Ser Ser Gly He Cys Gly He Ala He Asp Pro Leu Tyr Pro 195 200 205 Thr Leu Glu Glu 210 (2) INFORMATION FOR SEQ ID NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 335 amino acids (B) TYPE: amino acid •(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Met Trp Ala Thr Leu Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu Gly 1 5 10 15 Val Pro Val Cys Gly Ala Ala Glu Leu Ser Val Asn Ser Leu Glu Lys 20 25 30 Phe His Phe Lys Ser Trp Met Ser Lys His Arg Lys Thr Tyr Ser Thr 35 40 45 Glu Glu Tyr His His Arg Leu Gin Thr Phe Ala Ser Asn Trp Arg Lys 50 55 60 He Asn Ala His Asn Asn Gly Asn His Thr Phe Lys Met Ala Leu Asn 65 70 75 80 Gin Phe Ser Asp Met Ser Phe Ala Glu He Lys His Lys Tyr Leu Trp 85 90 95 Ser Glu Pro Gin Asn Cys Ser Ala Thr Lys Ser Asn Tyr Leu Arg Gly 100 105 110 Thr Gly Pro Tyr Pro Pro Ser Val Asp Trp Arg Lys Lys Gly Asn Phe 115 120 125 Val Ser Pro Val Lys Asn Gin Gly Ala Cys Gly Ser Cys Trp Thr Phe 130 135 140 Ser Thr Thr Gly Ala Leu Glu Ser Ala He Ala He Ala Thr Gly Lys 145 150 155 160 Met Leu Ser Leu Ala Glu Gin Gin Leu Val Asp Cys Ala Gin Asp Phe 165 170 175 Asn Asn Tyr Gly Cys Gin Gly Gly Leu Pro Ser Gin Ala Phe Glu Tyr 180 185 190 He Leu Tyr Asn Lys Gly He Met Gly Glu Asp Thr Tyr Pro Tyr Gin 195 200 205 Gly Lys Asp Gly Tyr Cys Lys Phe Gin Pro Gly Lys Ala He Gly Phe 210 215 220 Val Lys Asp Val Ala Asn He Thr He Tyr Asp Glu Glu Ala Met Val 225 230 235 240 Glu Ala Val Ala Leu Tyr Asn Pro Val Ser Phe Ala Phe Glu Val Thr 245 250 255 Gin Asp Phe Met Met Tyr Arg Thr Gly He Tyr Ser Ser Thr Ser Cys 260 265 270 His Lys Thr Pro Asp Lys Val Asn His Ala Val Leu Ala Val Gly Tyr 275 280 285 Gly Glu Lys Asn Gly He Pro Tyr Trp He Val Lys Asn Ser Trp Gly 290 295 300 Pro Gin Trp Gly Met Asn Gly Tyr Phe Leu He Glu Arg Gly Lys Asn 305 310 315 320 Met Cys Gly Leu Ala Ala Cys Ala Ser Tyr Pro He Pro Leu Val 325 330 335 (2) INFORMATION FOR SEQ ID NO:17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 339 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Met Trp Gin Leu Trp Ala Ser Leu Cys Cys Leu Leu Val Leu Ala Asn 1 5 10 15 Ala Arg Ser Arg Pro Ser Phe His Pro Val Ser Asp Glu Leu Val Asn 20 25 30 Tyr Val Asn Lys Arg Asn Thr Thr Trp Gin Ala Gly His Asn Phe Tyr 35 40 45 Asn Val Asp Met Ser Tyr Leu Lys Arg Leu Cys Gly Thr Phe Leu Gly 50 55 60 Gly Pro Lys Pro Pro Gin Arg Val Met Phe Thr Glu Asp Leu Lys Leu 65 70 75 80 Pro Ala Ser Phe Asp Ala Arg Glu Gin Trp Pro Gin Cys Pro Thr He . 85 90 95 Lys Glu He Arg Asp Gin Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly 100 105 110 Ala Val Glu Ala He Ser Asp Arg He Cys He His Thr Asn Ala His 115 120 125 Val Ser Val Glu Val Ser Ala Glu Asp Leu Leu Thr Cys Cys Gly Ser 130 135 140 Met Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pro Ala Glu Ala Trp Asn 145 150 155 160 Phe Trp Thr Arg Lys Gly Leu Val Ser Gly Gly Leu Tyr Glu Ser His 165 170 175 Val Gly Cys Arg Pro Tyr Ser He Pro Pro Cys Glu His His Val Asn 180 185 190 Gly S.er Arg Pro Pro Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Ser 195 200 205 Lys He Cys Glu Pro Gly Tyr Ser Pro Thr Tyr Lys Gin Asp Lys His 210 215 220 Tyr Gly Tyr Asn Ser Tyr Ser Val Ser Asn Ser Glu Lys Asp He Met 225 230 235 240 Ala Glu He Tyr Lys Asn Gly Pro Val Glu Gly Ala Phe Ser Val Tyr 245 250 255 Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val Tyr Gin His Val Thr Gly 260 265 270 Glu Met Met Gly Gly His Ala He Arg He Leu Gly Trp Gly Val Glu 275 280 285 Asn Gly Thr Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Thr Asp Trp 290 295 300 Gly Asp Asn Gly Phe Phe Lys He Leu Arg Gly Gin Asp His Cys Gly 305 310 315 320 He Glu Ser Glu Val Val Ala Gly He Pro Arg Thr Asp Gin Tyr Trp 325 330 335 Glu Lys He (2) INFORMATION FOR ' SEQ ID NO:18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 340 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: Met Leu Thr Ser He Leu Cys He Ala Ser Leu He Thr Phe Leu Glu 1 5 10 15 Ala His He Ser Val Lys Asn Glu Lys Phe Glu Pro Leu Ser Asp Asp 20 25 30 He He Ser Tyr He Asn Glu His Pro Asn Ala Gly Trp Arg Ala Glu 35 40 45 Lys Ser Asn Arg Phe His Ser Leu Asp Asp Ala Arg He Gin Met Gly 50 55 60 Ala Arg Arg Glu Glu Pro Asp Leu Arg Arg Lys Arg Arg Pro Thr Val 65 70 75 80 Asp His Asn Asp Trp Asn Val Glu He Pro Ser Asn Phe Asp Ser Arg 85 90 95 Lys Lys Trp Pro Gly Cys Lys Ser He Ala Thr He Arg Asp Gin Ser 100 105 110 Arg Cys Gly Ser Cys Trp Ser Phe Gly Ala Val Glu Ala Met Ser Asp 115 120 125 Arg Ser Cys He Gin Ser Gly Gly Lys Gin Asn Val Glu Leu Ser Ala 130 135 140 Val Asp Leu Leu Thr Cys Cys Glu Ser Cys Gly Leu Gly Cys Glu Gly 145 150 155 160 Gly He Leu Gly Pro Ala Trp Asp Tyr Trp Val Lys Glu Gly He Val 165 170 175 Thr Ala Ser Ser Lys Glu Asn His Thr Gly Cys Glu Pro Tyr Pro Phe 180 185 190 Pro Lys Cys Glu His His Thr Lys Gly Lys Tyr Pro Pro Cys Gly Ser 195 200 205 Lys He Tyr Asn Thr Pro Arg Cys Lys Gin Thr Cys Gin Arg Lys Tyr 210 215 220 Lys Thr Pro Tyr Thr Gin Asp Lys His Arg Gly Lys Ser Ser Tyr Asn 225 230 235 240 Val Lys Asn Asp Glu Lys Ala He Gin Lys Glu He Met Lys Tyr Gly 245 250 255 Pro Val Glu Ala Ser Phe Thr Val Tyr Glu Asp Phe Leu Asn Tyr Lys 260 265 270 Ser Gly He Tyr Lys His He Thr Gly Glu Ala Leu Gly Gly His Ala 275 280 285 Ile Arg He He Gly Trp Gly Val Glu Asn Lys Thr Pro Tyr Trp Leu 290 ' 295 300 He Ala Asn Ser Trp Asn Glu Asp Trp Gly Glu Asn Gly Tyr Phe Arg 305 310 315 320 He Val Arg Gly Arg Asp Glu Cys Ser He Glu Ser Glu Val He Ala 325 330 335 Gly Arg He Asn 340 (2) INFORMATION FOR SEQ ID NO:19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: ACAGCTCGAG GATCCGGCTG TACCCGACA 29 (2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: CTCGAGGATC CTATCACGGA AATCGTGC 28 (2) INFORMATION FOR SEQ ID N0:21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: Leu Pro Glu Ser Phe Asp Ala Arg Gin 1 5 (2) INFORMATION FOR SEQ ID NO:22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: Val Pro Ala Ser Phe Asp Ala Arg Gin 1 5 (2) INFORMATION FOR SEQ ID NO:23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: Gin Xaa Xaa Xaa Xaa Xaa Cys Trp Xaa Xaa Xaa 1 5 10 (2) INFORMATION FOR SEQ ID NO:24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Arg Ser Gin Trp Pro Gin Cys Trp Thr He Ser Glu He Arg Asp Gin 1 5 10 15 Ala Ser Cys Gly Ser Cys Trp Ala Ala Gly Gly Thr Ser Ala Met Ser 20 25 30 Asp Arg Val Cys He His Ser Asn Gly Gin Met Arg Pro Arg Leu Pro 35 40 45 Ala Ala Asp Pro Leu Ser Cys Cys Xaa Xaa Xaa Cys Gly Gin Gly Cys 50 55 60 Arg Val Gly Tyr His Arg Ala Xaa Trp Asp Tyr Trp Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Thr Pro Pro Cys 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu Glu Ala Cys Gin Thr Gly Tyr Asn 115 120 125 Lys Thr Tyr Glu Glu Asp Lys Phe Tyr Gly Asn Ser Ser Tyr Asn Val 130 135 140 Gly Asn Thr Glu Ser Xaa Xaa Xaa Xaa He Met Gin Glu He Lys Asn 145 150 155 160 Gly Pro Val Glu Val Thr Phe Ala Xaa He Phe Gin Asp Phe Gly Val 165 170 175 Tyr Arg Ser Gly He Tyr His His Val Ala Xaa Gly Lys Phe He Gly 180 185 190 Arg His Ala Val Arg Met He Gly Trp Gly Val Glu Asn Gly Val Asn 195 200 205 Tyr Trp Leu Met Ala Asn Ser Trp Asn Glu Glu Trp Gly Glu Asn Gly 210 215 220 Tyr Phe Arg Met Val Arg Gly Arg Asn Glu Thr 225 230 235

Claims: CLAIMS: 1. An isolated polypeptide comprising a sequence of amino acid residues wherein said sequence includes, in a contiguous sequence, amino acid residues: Gln-Xaa-Xaa-Xaa-Xaa Xaa-Cys-Trp-Xaa-Xaa-Xaa 2 wherein Xaa is any amino acid residue; Xaa j is Gly or Glu; and Xaa-, is Ser, Thr, Ala or Gly; said polypeptide further characterised by its ability to induce an immune response in a host against a helminth. 2. An isolated polypeptide according to claim 1 wherein said polypeptide is a cathepsin protease. 3. An isolated polypeptide according to claim 1 or 2 wherein said helminth is a trematode. 4. An isolated polypeptide according to claim 3 wherein said trematode is a species of Fasciola 5. An isolated polypeptide according to claim 4 wherein the trematode is Fasciola hepatica 6. An isolated polypeptide according to claim 4 or 5 wherein said polypeptide is derived from said species of Fasciola 7. An isolated polypeptide according to claim 6 wherein said polypeptide is derived from a Fasciola species in the mature state. 8. An isolated polypeptide according to claim 6 wherein said polypeptide is derived from a Fasciola in the newly excysted larval stage. 9. An isolated polypeptide according to claim 1 wherein said host is a mammal. 10. An isolated polypeptide according to claim 9 wherein said mammal is a livestock animal. 11. An isolated polypeptide according to claim 10 wherein said livestock animal is an ovine or bovine species. 12. An isolated polypeptide according to claim 1 or 11 wherein the immune response is a protective immune response. 13. An isolated polypeptide according to claim 12 wherein said immune response is a humoral immune response. 14. An isolated polypeptide according to claim 2 wherein said polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO. 2. 15. An isolated polypeptide according to claim 2 wherein said polypeptide comprises an amino acid sequence substantially as set forth in SEQ ID NO. 12. 16. An isolated polypeptide according to claim 2 wherein said polypeptide comprises an N-terminal sequence substantially as set forth in SEQ ID NO. 21 and 22. 17. An isolated polypeptide according to claim 1 or 14 or 15 or 16 in recombinant form. 18. An antigenic fragment, part, derivative or analogue of a polypeptide according to claim 1 or 14 or 15 or 16. 19. A polypeptide which: (i) is a cathepsin protease or like molecule; (ii) comprises a sequence of amino acids which includes the contiguous amino acid sequence Gln-Xaa-Xaa-Xaa-Xaa Xaa-Cys-Trp-Xaa-Xaa- Xaa 2 (iii) is isolatable from a helminth; and (iv) comprises an amino acid sequence having at least 50% amino acid sequence identity to all or part of the amino acid sequence substantially as set forth in SEQ ID NO. 2 or SEQ ID NO. 12 or SEQ ID NO. 24. 20. An isolated polypeptide according to claim 19 wherein the helminth is a trematode. 21. An isolated polypeptide according to claim 20 wherein the trematode is a species of Fasciola 22. An isolated polypeptide according to claim 20 wherein the trematode is Fasciola hepatica 23. An isolated polypeptide according to claim 20 wherein the trematode is Fasciola gigantica. 24. An isolated polypeptide according to claim 22 wherein the Fasciola species is in the mature state. 25. An isolated polypeptide according to claim 22 wherein said polypeptide is derived from a Fasciola in the newly excysted larval stage. 26. An isolated polypeptide according to claim 19 further characterised by being capable of inducing an immune response in a host. 27. An isolated polypeptide according to claim 26 wherein the host is a mammal. 28. An isolated polypeptide according to claim 27 wherein the mammal is livestock animal. 29. An isolated polypeptide according to claim 28 wherein the livestock anima is an ovine or bovine species. 30. An isolated polypeptide according to claim 29 wherein the immune response is a protective immune response against helminth infection. 31. An isolated or recombinant polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO. 2. 32. An isolated or recombinant polypeptide having an amino acid sequence substantially as set forth in SEQ ID NO. 12 or SEQ ID NO. 24. 33. An isolated or recombinant polypeptide having an N-terminal amino acid sequence substantially as set forth in SEQ ID NO. 21 or SEQ ID NO. 22. 34. A nucleic acid molecule comprising a sequence of nucleotides which: (i) encodes a cathepsin protease; (ii) is isolatable from a helminth species; and (iii) hybridises under low stringency conditions to all or part of the nucleic acid sequence set forth in SEQ ID NO. 1 or 11 or to a complementary form thereof. 35. A nucleic acid molecule according to claim 34 wherein the helminth is a trematode. 36. A nucleic acid molecule according to claim 35 wherein the trematode is a Fasciola species. 37. A nucleic acid molecule according to claim 36 wherein the Fasciola specie is Fasciola hepatica. 38. A nucleic acid molecule according to claim 36 wherein the Fasciola specie is in a mature state. 39. A nucleic acid molecule according to claim 36 wherein the Fasciola specie is in a newly excysted larval stage. 40. A nucleic acid molecule having a sequence of nucleotides substantially as se forth in SEQ ID NO. 1. 41. A nucleic acid molecule having a sequence of nucleotides substantially as se forth, in SEQ ID NO. 11. 42. A method for reducing spread of a helminth parasite, said method comprisin administering to an animal susceptible to infection with said parasite a effective amount of a polypeptide derived from or a comprising cathepsi protease according to claim 1 or 19 for a time and under conditions sufficien for an immune response to develop to said cathepsin protease. 43. A method according to claim 43 wherein the helminth is a trematode. 44. A method according to claim 42 wherein the immune response is a protectiv immune response. 45. A method according to claim 43 wherein the species of Fasciola is in matur form. 46. A method according to claim 43 wherein the species of Fasciola is a newl excysted larval stage. 47. A method according to claim 43 wherein the helminth is a species of Fasciola 48. A method according to claim 47 wherein the Fasciola species is Fasciol hepatica 49. A method according to claim 42 wherein the host is a mammal. 50. A method according to claim 42 wherein the mammal is a livestock animal. 51. A method according to claim 50 wherein the livestock animal is an ovine or bovine species. 52. A vaccine composition comprising a polypeptide according to claim 1 or 19 and one or more carriers and/or diluents acceptable for veterinary use.

Inventors: MILNER ANDREW RICHARD, PANACCIO MICHEL, SPITHILL TERENCE WILLIAM, WIJFFELS GENE LOUISE, MILNER, ANDREW, RICHARD, PANACCIO, MICHEL, SPITHILL, TERENCE, WILLIAM, WIJFFELS, GENE, LOUISE
Applicants: DARATECH PTY LTD, MILNER ANDREW RICHARD, PANACCIO MICHEL, SPITHILL TERENCE WILLIAM, WIJFFELS GENE LOUISE, DARATECH PTY. LTD., MILNER, ANDREW, RICHARD, PANACCIO, MICHEL, SPITHILL, TERENCE, WILLIAM, WIJFFELS, GENE, LOUISE
Application year: 1994
Main classification: A61K37
IPC codes: A61K38, A61K39, A61P33, C12N9, C12N15